Combination therapy using c-met inhibitor and igf-1r inhibitor

ABSTRACT

A method of preventing and/or treating a cancer, the method including co-administering a dual inhibitor of c-Met (hereinafter, ‘c-Met inhibitor’) and an IGF-1R inhibitor to a subject in need thereof and a use of IGF-1R as a marker for resistance to a c-Met inhibitor.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of Korean Patent Application No. 10-2014-0163792 filed on Nov. 21, 2014 in the Korean Intellectual Property Office, the entire disclosure of which is hereby incorporated by reference.

INCORPORATION-BY-REFERENCE OF MATERIAL ELECTRONICALLY SUBMITTED

Incorporated by reference in its entirety herein is a computer-readable nucleotide/amino acid sequence listing submitted herewith and identified as follows: One 143,800 byte ASCII (Text) file named “721848_ST25.TXT,” created on Nov. 23, 2015.

BACKGROUND OF THE INVENTION

1. Field

Provided is a method of preventing and/or treating a cancer, the method including co-administering a c-Met inhibitor and an IGF-1R inhibitor to a subject in need thereof and a use of IGF-1R as a marker for resistance to a c-Met inhibitor.

2. Description of the Related Art

It has been shown that resistance to a drug having a specific target is more common compared to resistance to a drug having no specific target. In addition, it is also known that the indications on which a targeting drug has a therapeutic effect when it is treated alone are limited. Therefore, co-administration of two or more targeting drugs can maximize the therapeutic effect in a subject by overcoming resistance caused by exclusive treatment with only one of the targeting drugs, and by exhibiting a therapeutic effect for an indication in which one of the targeting drugs has no therapeutic effect. Such co-administration is expected to contribute to extending the scope of indications to be treated by target drugs and to overcoming resistance thereto.

Therefore, for more effective treatment of a disease, there remains a need to develop effective combination therapy targeting two or more targets.

BRIEF SUMMARY OF THE INVENTION

An embodiment provides a combination therapy targeting c-Met and IGF-1R.

Another embodiment provides a pharmaceutical composition for combination therapy for treating and/or preventing cancer, the composition including a c-Met inhibitor and an IGF-1R inhibitor, as active ingredients.

Another embodiment provides a kit for treating and/or preventing cancer, including a first pharmaceutical composition including a c-Met inhibitor, a second pharmaceutical composition including an IGF-1R inhibitor, and a package container.

Another embodiment provides a method of treating and/or preventing cancer, the method including co-administering a c-Met inhibitor and an IGF-1R inhibitor to a subject in need thereof.

Another embodiment provides a pharmaceutical composition for overcoming resistance to a c-Met inhibitor, the composition including an IGF-1R inhibitor.

Another embodiment provides a method of overcoming resistance to a c-Met inhibitor, the method including administering an IGF-1R inhibitor together with a c-Met inhibitor to a subject who has resistance to the c-Met inhibitor.

Another embodiment provides a marker for predicting and/or examining (monitoring) an effect of a c-Met inhibitor, including IGF-1R protein and/or nucleic acids (e.g., full-length DNA, cDNA or mRNA) encoding IGF-1R. The marker may be used for determining the presence or absence of resistance to a c-Met inhibitor.

Another embodiment provides a method for predicting and/or examining (monitoring) an effect of a c-Met inhibitor, including measuring a level of IGF-1R protein and/or nucleic acids (e.g., full-length DNA, cDNA or mRNA) encoding IGF-1R in a biological sample obtained from a subject. The method may be used for determining the presence or absence of resistance to a c-Met inhibitor.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 displays immunoblotting results showing expression levels of receptor tyrosine kinases (RTKs; EGFR, ErbB2, HGFR (c-Met), and IGF-1R) in Hs746T and SNU-5 cell lines (anti-c-Met antibody sensitive) and SNU-668 and YCC-11 cell lines (anti-c-Met antibody resistant), wherein the lowest image is a reference indicating the positions of proteins.

FIG. 2 is schematic view of the results of FIG. 1, wherein the degree of darkness is directly proportional to the level of expression.

FIG. 3 is a graph showing the expression level of c-Met from the immunoblotting results shown in FIG. 1, wherein the expression level is obtained by normalizing the results (darkness and size of dots) shown in FIG. 1 with a positive control dot (which corresponds to a protein present on all membranes regardless of dose of antibody, and is used for normalizing RTKs expression to compare the expression between sample and membrane; loading control), and quantifying the relative value (%; intensity; mean pixel density) of darkness and size of c-Met corresponding dot comparing to that of the positive control dot (100%).

FIG. 4 is a graph showing the expression level of IGF-1R from the immunoblotting results shown in FIG. 1, wherein the expression level is obtained by normalizing the results (darkness and size of dots) shown in FIG. 1 with a positive control dot, and quantifying the relative value (%; intensity; mean pixel density) of darkness and size of IGF-1R corresponding dot comparing to that of the positive control dot (100%).

FIG. 5 is a graph showing the expression level of EGFR from the immunoblotting results shown in FIG. 1, wherein the expression level is obtained by normalizing the results (darkness and size of dots) shown in FIG. 1 with a positive control dot, and quantifying the relative value (%; intensity; mean pixel density) of darkness and size of EGFR corresponding dot comparing to that of the positive control dot (100%).

FIG. 6 is a graph showing the expression level of ErbB2 from the immunoblotting results shown in FIG. 1, wherein the expression level is obtained by normalizing the results (darkness and size of dots) shown in FIG. 1 with a positive control dot, and quantifying the relative value (%; intensity; mean pixel density) of darkness and size of ErbB2 corresponding dot comparing to that of the positive control dot (100%).

FIG. 7 is a graph showing cell proliferation inhibition effects of administration of anti-c-Met antibody L3-1Y/IgG2 alone, IGF-1R inhibitor linsitinib alone, and a combination of L3-1Y/IgG2 and linsitinib, in anti-c-Met antibody resistance induced cell line MKN45 re#1.

FIG. 8 is a graph showing cell proliferation inhibition effects of administration of anti-c-Met antibody L3-1Y/IgG2 alone, IGF-1R inhibitor linsitinib alone, and a combination of L3-1Y/IgG2 and linsitinib, in anti-c-Met antibody resistance induced cell line MKN45 re#24.

FIG. 9 is a graph showing the concentration dependent cell proliferation inhibition effect of anti-c-Met antibody L3-1Y/IgG2 in anti-c-Met antibody resistance induced cell lines MKN45 re#1 and MKN45 re#24, and non-resistant cell line MKN45.

DETAILED DESCRIPTION OF THE INVENTION

A c-Met inhibitor, such as an anti-c-Met antibody, generally exhibits strong anticancer effects on cancers having a high expression level of c-Met, such as gastric cancer, lung cancer, and the like. However, when resistance to an anti-c-Met antibody is inherently present in a subject (e.g., a c-Met resistant cancer cell) or acquired by repeated administration of the anti-c-Met antibody, the anti-c-Met antibody cannot exhibit its effect on the subject, even if the expression level of c-Met in the subject is high. In cells with a high level of c-Met expression which are resistant to an anti-c-Met antibody (e.g., does not exhibit its anticancer effect), the expression level of IGF-1R is considerably higher compared to cells on which an anti-c-Met antibody exhibits its effect (e.g., anticancer effect). Based thereon, IGF-1R can serve as a marker for determining the presence or absence of resistance to an anti-c-Met antibody and/or as a target for a drug for being co-administered together with an anti-c-Met antibody in a combination therapy.

By co-administering an anti-c-Met antibody and an IGFR1 inhibitor to simultaneously inhibit c-Met and IGF-1R, an anticancer effect can be achieved even in cases where such an effect cannot be obtained when only one of the targets is inhibited. Thus, the applicable scope of an anti-c-Met antibody can be expanded and resistance to an anti-c-Met antibody can be overcome.

Herein, the term “co-administration” and “combined administration” may be used interchangeably (i.e., have the same meaning).

The “c-Met” or “c-Met proteins” refer to receptor tyrosine kinases that bind to hepatocyte growth factors (HGF). The c-Met proteins may be those derived from all kinds of species, particularly a mammal, for example, those derived from a primate such as human c-Met (e.g. NP_000236.2), monkey c-Met (e.g., Macaca mulatta, NP_001162100), and the like, or those derived from a rodent such as mouse c-Met (e.g., NP_032617.2), rat c-Met (e.g., NP_113705.1), and the like. These proteins may include, for example, polypeptides encoded by the nucleotide sequence identified as GenBank Accession Number NM_000245.2, or proteins encoded by the polypeptide sequence identified as GenBank Accession Number NM_000236.2, or extracellular domains thereof. The receptor tyrosine kinase c-Met is involved in several mechanisms including cancer incidence, cancer metastasis, cancer cell migration, cancer cell penetration, angiogenesis, etc.

IGF-1R (insulin-like growth factor 1 receptor) binds to IGF1 (insulin-like growth factor 1) or IGF2 (insulin-like growth factor 2), and binds to insulin at a low affinity. The binding between IGF1 and IGF-1R increases the activity of receptor tyrosine kinases and induces auto-phosphorylation of the receptors or phosphorylation of internal related materials, thereby performing signal transduction. IGF-1R promotes proliferation of normal cells, but it can cause abnormal growth of cells. The overexpression of IGF-1R may cause formation of neoplasm, and thus its importance in cancer therapy emerged. The IGF-1R protein may be obtained from any species; for example, it may be at least one selected from the group consisting of human IGF-1R (e.g., AAI43722.1 (coding gene (cDNA or mRNA): BC143721.1), NP_000866.1 (coding gene (cDNA or mRNA): NM_000875.4), NP_001278787.1 (coding gene (cDNA or mRNA): NM_001291858.1)), etc.), mouse IGF-1R (e.g., AAI38870.1 (coding gene (cDNA or mRNA): BC138869.1), AAI38869.1 (coding gene (cDNA or mRNA): BC138868.1), NP_034643.2 (coding gene (cDNA or mRNA): NM_010513.2), etc.), and the like, but not limited thereto.

It has been shown that IGF-1R signaling induces resistance to cytotoxic therapy and has a high relation with resistance to EGFR-targeting therapy. In particular, high expression of IGF-1R from cancer cells surgically excised from a gastric cancer patent is related to poor results of cancer therapy. In 86 gastric cancer patients having the surgical operation, the survival time is relatively longer for patients with low expression levels of IGF-1R and EGFR in the excised gastric cancer tissue compared to patients with high expression levels of IGF-1R and EGFR. Therefore, IGF-1R is considered as one of the important targets contributing to cancer cell growth through cross-talk with other RTKs.

As used herein, the term “c-Met inhibitor” may refer to any agent (e.g., a compound or composition) capable of inhibiting activity and/or expression of c-Met, or blocking c-Met signaling by inhibiting ligands thereof, thereby preventing, improving, alleviating, reducing, and/or treating a c-Met associated disease (e.g., a disease associating with abnormal activation and/or overexpression of c-Met, such as cancer) or its symptom. In addition, the c-Met inhibitor may refer to any substance capable of recognizing and/or binding to c-Met to degrade and/or inhibit the functions or expression thereof. For example, the c-Met inhibitor may be an anti-c-Met antibody which recognizes and/or binds to c-Met. The anti-c-Met antibody may bind to c-Met and induce internalization (into a cell) and degradation of c-Met.

As used herein, the term “effect of the c-Met inhibitor” may refer to an effect to prevent, improve, alleviate, reduce the severity of, and/or otherwise treat a c-Met associated disease, such as a cancer. For example, the effect of the c-Met inhibitor may refer to an effect of reducing (or inhibiting proliferation of) cancer cells or cancer tissues, killing cancer cells or cancer tissues, and/or inhibiting invasion and/or migration of cancer cells related to metastasis. Thus, the effect may refer to an anticancer effect such as inhibiting cancer cell proliferation, migration (metastasis), or invasion, or inducing cancer cell apoptosis, etc.

The present disclosure suggests a combination therapy by combined administration of a c-Met inhibitor and an IGF-1R inhibitor, having advantages to expand the scope of application of the c-Met inhibitor to indications (diseases) on which the c-Met inhibitor does not exhibit an effect when used alone, and overcome resistance to a c-Met inhibitor. The combined administration of the c-Met inhibitor and the IGF-1R inhibitor also leads to more effective treatment of a disease (e.g., cancer) upon which a c-Met inhibitor and/or IGF-1R inhibitor exhibits the effect even when each of them if used alone, i.e., a disease that is not resistant to a c-Met inhibitor. Also, by such combined administration, it is believed that the dose of each inhibitor can be reduced and/or the administration interval can be increase (i.e., the frequency of administration can be decreased), thereby reducing side effects in a subject to be administered with the inhibitors.

One embodiment provides a pharmaceutical composition for combination therapy for preventing and/or treating of a cancer, comprising or consisting essentially of a c-Met inhibitor and an IGF-1R inhibitor as active ingredients.

The pharmaceutical composition for combination therapy may be a mixed formulation (e.g., a single composition comprising two active ingredients) of a c-Met inhibitor and an IGF-1R inhibitor. The IGF-1R inhibitor and c-Met inhibitor may be included in any amount that is pharmaceutically effective when used together. The composition thus formulated can be used for simultaneous administration of the two active ingredients.

Alternatively, each of the c-Met inhibitor and the IGF-1R inhibitor can be formulated in a separate composition and the two active ingredients (or compositions) can be separately administered simultaneously or sequentially. For instance, a first pharmaceutical composition including a pharmaceutically effective amount of the IGF-1R inhibitor as an active ingredient and a second pharmaceutical composition including a pharmaceutically effective amount of the c-Met inhibitor as an active ingredient can be administered simultaneously or sequentially. In the case of the sequential administration, any order of administration may be used.

Another embodiment provides a kit useful for preventing and/or treating a cancer, comprising or consisting essentially of a first pharmaceutical composition including an IGF-1R inhibitor as an active ingredient, a second pharmaceutical composition including a c-Met inhibitor as an active ingredient, and a package container. The IGF-1R inhibitor and c-Met inhibitor may be used in amounts that are pharmaceutically effective when combined, which amount may be determined by the skilled medical practitioner or medical researcher. The package container can be any container that holds or otherwise links the two compositions in individual containers together in a single unit (e.g., a box that holds both containers, or plastic wrap that binds both containers together), or the package container may be a single, divided container having at least two chambers that each hold one of the two compositions.

A method of combination therapy for preventing and/or treating a cancer also is provided. The method comprises or consists essentially of co-administering a c-Met inhibitor and an IGF-1R inhibitor to a subject in need of the prevention and/or treatment of cancer. The IGF-1R inhibitor and c-Met inhibitor may be administered in amounts that are pharmaceutically effective when combined, which amount may be determined by the skilled medical practitioner or medical researcher. The method may further include, prior to the co-administration step, a step of identifying a subject in need of the prevention and/or treatment of cancer. The step of identifying may be conducted by any manners and/or methods known to the relevant field for identifying whether or not a subject needs the prevention and/or treatment of cancer. For example, the step of identifying may include diagnosing a subject to have a cancer, or identifying a subject who is diagnosed as a cancer subject, particularly a cancer associated with c-Met expression or a cancer exhibiting resistance to treatment with a c-Met inhibitor alone.

Co-administration may be conducted by administering the two active ingredients (i.e., c-Met inhibitor and IGF-1R inhibitor) simultaneously; for example, by administering a mixed formulation (e.g., single composition) of a c-Met inhibitor and an IGF-1R inhibitor, as described herein. Alternatively, the c-Met inhibitor and IGF-1R inhibitor can be administered separately. The co-administration may be conducted by a first step of administering a c-Met inhibitor, and a second step of administering an IGF-1R inhibitor, wherein the first and the second administration steps may be conducted simultaneously or sequentially. In case of the sequential administration, the first step and the second step may be performed in any order. In the sequential administration the first step and the second step may be separated by any suitable time interval (about 1 second to about 24 hours, about 1 second to about 12 hours, or about 1 second to about 60 minutes (e.g., about 1 second to about 10 minutes); or 1-60 seconds, 1-60 minutes, 1-24 hours, or 1-7 days, 1-14 days, 7-14 days, 1-30 days, or so on). The c-Met inhibitor and IGF-1R inhibitor may be administered in amounts that are pharmaceutically effective when combined, which amount may be determined by the skilled medical practitioner or medical researcher.

The term “the pharmaceutically effective amount” as used in this specification refers to an amount of which each active ingredient can exert pharmaceutically significant effects (e.g., an amount sufficient to prevent or treat cancer in a subject).

The subject for administration may be any animal, for example a mammal including a primate such as a human or a monkey, or a rodent such as a mouse or a rat, or a cell or tissue separated therefrom, or a culture of the cell or tissue. In particular, the subject may be a mammal including a primate such as a human or a monkey, or a rodent such as a mouse or a rat, or a cell or tissue separated therefrom, or a culture of the cell or tissue, who has an innate resistance or acquired resistance to a c-Met inhibitor when used alone.

By combined administration of a c-Met inhibitor and an IGF-1R inhibitor, a synergistic therapeutic effect can be achieved compared to administration of c-Met only or IGF-1R only. Furthermore, the increased therapeutic effect from the combined administration can be obtained even for a disease (e.g., a cancer) in which each of the inhibitors does not exhibit its effect or which has resistance to each of the inhibitors.

The IGF-1R inhibitor may refer to any drug capable of targeting and/or inhibiting IGF-1R protein or a nucleic acid (e.g., full-length DNA, cDNA or mRNA) encoding IGF-1R. In particular, the IGF-1R inhibitor may be at least one selected from the group consisting of a compound (small molecular compound or its pharmaceutically acceptable salt), a protein (e.g., an antibody, an antigen-binding fragment of antibody, etc.), a nucleic acid (e.g., an aptamer, siRNA, shRNA, microRNA, etc.) and the like, which targets and/or inhibit IGF-1R protein and/or nucleic acids (e.g., full-length DNA, cDNA or mRNA) encoding IGF-1R protein For example, the IGF-1R inhibitor may be at least one selected from the group consisting of linsitinib(OSI-906; 3-[8-Amino-1-(2-phenyl-7-quinolyl)imidazo[1,5-a]pyrazin-3-yl]-1-methyl-cyclobutanol), NVP-AEW541 (CAS#475489-16-8; 7-((1s,3s)-3-(azetidin-1-ylmethyl)cyclobutyl)-5-(3-(benzyloxy)phenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine), GSK1904529A (CAS#1089283-49-7; N-(2,6-difluorophenyl)-5-(3-(2-(5-ethyl-2-methoxy-4-(4-(4-(methylsulfonyl)piperazin-1-yl)piperidin-1-yl)phenylamino)pyrimidin-4-yl)H-imidazo[1,2-a]pyridin-2-yl)-2-methoxybenzamide), NVP-ADW742(CAS#475488-23-4; 5-(3-(benzyloxy)phenyl)-7-((1r,3r)-3-(pyrrolidin-1-ylmethyl)cyclobutyl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine), BMS-536924(CAS#468740-43-4; 4-[[(2S)-2-(3-chlorophenyl)-2-hydroxyethyl]amino]-3-[4-methyl-6-(4-morpholinyl)-1H-benzimidazol-2-yl]-2(1H)-pyridinone), figitumumab (CP-751,871), cixutumumab (IMC-A12), dalotuzumab (MK-0646), R1507 (fully human anti-IGF-1R antibody), XL-228 (CAS#898280-07-4), INSM-18 (nordihydroguaiaretic acid; NDGA; 4,4′-((2R,3S)-2,3-dimethylbutane-1,4-diyl)bis(benzene-1,2-diol)), BMS-754807 ((S)-1-(4-((5-cyclopropyl-1H-pyrazol-3-yl)amino)pyrrolo[2,1-f][1,2,4]triazin-2-yl)-N-(6-fluoropyridin-3-yl)-2-methylpyrrolidine-2-carboxamide), AG-1024 (Tyrphostin; CAS#65678-07-1; 2-(3-bromo-5-tert-butyl-4-hydroxybenzylidene)malononitrile), GSK1838705A (CAS#1116235-97-2; 2-(2-(1-(2-(dimethylamino)acetyl)-5-methoxyindolin-6-ylamino)-7H-pyrrolo[2,3-d]pyrimidin-4-ylamino)-6-fluoro-N-methylbenzamide), PQ 401 (CAS#196868-63-0; N-(5-Chloro-2-methoxyphenyl)-N′-(2-methyl-4-quinolinyl)urea), and pharmaceutically acceptable salts thereof, but not be limited thereto.

XL-228:

The c-Met inhibitor may be any drug targeting c-Met protein and/or nucleic acids (e.g., full-length DNA, cDNA or mRNA) encoding c-Met. In particular, c-Met inhibitor may be at least one selected from the group consisting of an antibody or an antigen-binding fragment thereof, an aptamer, siRNA, shRNA, microRNA, a compound (e.g., a small molecular compound or a pharmaceutically acceptable salt thereof), and the like, against (binding to or otherwise inhibiting) c-Met and/or a nucleic acid (e.g., full-length DNA, cDNA or mRNA) encoding c-Met.

For example, the c-Met inhibitor may be at least one selected from the group consisting of an anti-c-Met antibody or an antigen-binding fragment thereof, detailed below, crizotinib (PF-02341066; PF-02341066; 3-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-5-(1-piperidin-4-ylpyrazol-4-yl)pyridin-2-amine), cabozantinib (XL-184; N-(4-(6,7-dimethoxyquinolin-4-yloxy)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide), foretinib (N-(3-fluoro-4-(6-methoxy-7-(3-morpholinopropoxy)quinolin-4-yloxy)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide), PHA-665752((R,Z)-5-(2,6-dichlorobenzylsulfonyl)-3-((3,5-dimethyl-4-(2-(pyrrolidin-1-ylmethyl)pyrrolidine-1-carbonyl)-1H-pyrrol-2-yl)methylene)indolin-2-one), SU11274((Z)—N-(3-chlorophenyl)-3-((3,5-dimethyl-4-(1-methylpiperazine-4-carbonyl)-1H-pyrrol-2-yl)methylene)-N-methyl-2-oxoindoline-5-sulfonamide), SGX-523 (6-(6-(1-methyl-1H-pyrazol-4-yl)-[1,2,4]triazolo[4,3-1)]pyridazin-3-ylthio)quinoline), PF-04217903 (2-(4-(3-(quinolin-6-ylmethyl)-3H-[1,2,3]triazolo[4,5-b]pyrazin-5-yl)-1H-pyrazol-1-yl)ethanol), EMD 1214063(Benzonitrile, 3-[1,6-Dihydro-1-[[3-[5-[(1-Methyl-4-Piperidinyl)Methoxy]-2-PyriMidinyl]Phenyl]Methyl]-6-Oxo-3-Pyridazinyl]), golvatinib (N-(2-fluoro-4-((2-(4-(4-methylpiperazin-1-yl)piperidine-1-carboxamido)pyridin-4-yl)oxy)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide), INCB28060(2-fluoro-N-methyl-4-(7-(quinolin-6-ylmethyl)imidazo[1,2-b)][1,2,4]triazin-2-yl)benzamide), MK-2461 (N-((2R)-1,4-Dioxan-2-ylmethyl)-N-methyl-N′-[3-(1-methyl-1H-pyrazol-4-yl)-5-oxo-5H-benzo[4,5]cyclohepta[1,2-b]pyridin-7-yl]sulfamide), tivantinib (ARQ 197; (3R,4R)-3-(5,6-Dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione), NVP-BVU972(6-[[6-(1-Methyl-1H-pyrazol-4-yl)imidazo[1,2-b]pyridazin-3-yl]methyl]quinoline), AMG458({1-(2-hydroxy-2-methylpropyl)-N-[5-(7-methoxyquinolin-4-yloxy)pyridin-2-yl]-5-methyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazole-4-carboxamide}), BMS 794833 (N-(4-((2-amino-3-chloropyridin-4-yl)oxy)-3-fluorophenyl)-5-(4-fluorophenyl)-4-oxo-1,4-dihydropyridine-3-carboxamide), BMS 777607(N-[4-[(2-Amino-3-chloropyridin-4-yl)oxy]-3-fluorophenyl]-4-ethoxy-1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxamide), MGCD-265 (N-(3-Fluoro-4-(2-(1-methyl-1H-imidazol-4-yl)thieno[3,2-b]pyridin-7-yloxy)phenylcarbamothioyl)-2-phenylacetamide), AMG-208(7-Methoxy-4-[(6-phenyl-1,2,4-triazolo[4,3-b]pyridazin-3-yl)methoxy]quinoline), BMS-754807((2S)-1-[4-[(5-Cyclopropyl-1H-pyrazol-3-yl)amino]pyrrolo[2,1-f][1,2,4]triazin-2-yl]-N-(6-fluoro-3-pyridinyl)-2-methyl-2-pyrrolidinecarboxamide), JNJ-38877605(6-[Difluoro[6-(1-methyl-1H-pyrazol-4-yl)-1,2,4-triazolo[4,3-b]pyridazin-3-yl]methyl]quinoline), and pharmaceutically acceptable salts thereof.

The anti-c-Met antibody or an antigen-binding fragment thereof may be any type of antibody or antigen-binding fragment thereof, which is capable of specifically recognizing and/binding to c-Met. The antigen-binding fragment may be, for example, scFv, (scFv)2, scFv-Fc, Fab, Fab′ or F(ab′)2.

In one embodiment, the anti-c-Met antibody may be any antibody or antigen-binding fragment that acts on c-Met to induce intracellular internalization and degradation of c-Met. In another embodiment, the anti-c-Met antibody may be an antibody recognizing a specific region of c-Met, e.g., a region in the SEMA domain, as an epitope.

c-Met, a receptor for hepatocyte growth factor (HGF), may be divided into three portions: extracellular, transmembrane, and intracellular. The extracellular portion is composed of an α-subunit and a β-subunit which are linked to each other through a disulfide bond, and contains a SEMA domain responsible for binding HGF, a PSI domain (plexin-semaphorin-integrin homology domain) and an IPT domain (immunoglobulin-like fold shared by plexins and transcriptional factors domain). The SEMA domain of c-Met protein may have the amino acid sequence of SEQ ID NO: 79, and is an extracellular domain that functions to bind HGF. A specific region of the SEMA domain, that is, a region having the amino acid sequence of SEQ ID NO: 71, which corresponds to a range from amino acid residues 106 to 124 of the amino acid sequence of the SEMA domain (SEQ ID NO: 79) of c-Met protein, is a loop region between the second and the third propellers within the epitopes of the SEMA domain. The anti-c-Met antibody used in accordance with the composition and method described herein may be an antibody that binds this region acts as an epitope.

The term “epitope” as used herein, refers to an antigenic determinant, a part of an antigen recognized by an antibody. In one embodiment, the epitope may be a region including about 5 or more contiguous amino acid residues within the SEMA domain (SEQ ID NO: 79) of c-Met protein, for instance, about 5 to about 19 contiguous amino acid residues within the amino acid sequence of SEQ ID NO: 71. For example, the epitope may be a polypeptide having about 5 to about 19 contiguous amino acids selected from among partial combinations of the amino acid sequence of SEQ ID NO: 71, wherein the polypeptide essentially includes the amino sequence of SEQ ID NO: 73 (EEPSQ) serving as an essential element for the epitope. For example, the epitope may be a polypeptide including, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 71, SEQ ID NO: 72, or SEQ ID NO: 73.

The epitope including the amino acid sequence of SEQ ID NO: 72 corresponds to the outermost part of the loop between the second and third propellers within the SEMA domain of a c-Met protein. The epitope including the amino acid sequence of SEQ ID NO: 73 is a site to which the antibody or antigen-binding fragment according to one embodiment most specifically binds.

Thus, the anti-c-Met antibody may specifically bind to an epitope which has about 5 to about 19 contiguous amino acids selected from among partial combinations of the amino acid sequence of SEQ ID NO: 71, including SEQ ID NO: 73 as an essential element. For example, the anti-c-Met antibody may specifically bind to an epitope including the amino acid sequence of SEQ ID NO: 71, SEQ ID NO: 72, or SEQ ID NO: 73.

In one embodiment, the anti-c-Met antibody or an antigen-binding fragment thereof may comprise or consist essentially of:

at least one heavy chain complementarity determining region (CDR) selected from the group consisting of (a) a CDR-H1 including the amino acid sequence of SEQ ID NO: 4; (b) a CDR-H2 including the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 2, or an amino acid sequence having about 8-19 consecutive amino acids within SEQ ID NO: 2 including amino acid residues from the 3^(rd) to 10^(th) positions of SEQ ID NO: 2; and (c) a CDR-H3 including the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 85, or an amino acid sequence having about 6-13 consecutive amino acids within SEQ ID NO: 85 including amino acid residues from the 1^(st) to 6^(th) positions of SEQ ID NO: 85, or a heavy chain variable region including the at least one heavy chain complementarity determining region;

at least one light chain complementarity determining region (CDR) selected from the group consisting of (a) a CDR-L1 including the amino acid sequence of SEQ ID NO: 7, (b) a CDR-L2 including the amino acid sequence of SEQ ID NO: 8, and (c) a CDR-L3 including the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 15, SEQ ID NO: 86, or an amino acid sequence having 9-17 consecutive amino acids within SEQ ID NO: 89 including amino acid residues from the 1^(st) to 9^(th) positions of SEQ ID NO: 89, or a light chain variable region including the at least one light chain complementarity determining region;

a combination of the at least one heavy chain complementarity determining region and at least one light chain complementarity determining region; or

a combination of the heavy chain variable region and the light chain variable region.

Herein, the amino acid sequences of SEQ ID NOS: 4 to 9 are respectively represented by following Formulas I to VI, below:

Formula I (SEQ ID NO: 4) Xaa₁-Xaa₂-Tyr-Tyr-Met-Ser,

wherein Xaa₁ is absent or Pro or Ser, and Xaa₂ is Glu or Asp,

Formula II (SEQ ID NO: 5) Arg-Asn-Xaa₃-Xaa₄-Asn-Gly-Xaa₅-Thr,

wherein Xaa₃ is Asn or Lys, Xaa₄ is Ala or Val, and Xaa₅ is Asn or Thr,

Formula III (SEQ ID NO: 6) Asp-Asn-Trp-Leu-Xaa₆-Tyr,

wherein Xaa₆ is Ser or Thr,

Formula IV (SEQ ID NO: 7) Lys-Ser-Ser-Xaa₇-Ser-Leu-Leu-Ala-Xaa₈-Gly-Asn- Xaa₉-Xaa₁₀-Asn-Tyr-Leu-Ala 

wherein Xaa₇ is His, Arg, Gln, or Lys, Xaa₈ is Ser or Trp, Xaa₉ is His or Gln, and Xaa₁₀ is Lys or Asn,

Formula V (SEQ ID NO: 8) Trp-Xaa₁₁-Ser-Xaa₁₂-Arg-Val-Xaa₁₃ 

wherein Xaa₁₁ is Ala or Gly, Xaa₁₂ is Thr or Lys, and Xaa₁₃ is Ser or Pro, and

Formula VI (SEQ ID NO: 9) Xaa₁₄-Gln-Ser-Tyr-Ser-Xaa₁₅-Pro-Xaa₁₆-Thr 

wherein Xaa₁₄ is Gly, Ala, or Gln, Xaa₁₅ is Arg, His, Ser, Ala, Gly, or Lys, and Xaa₁₆ is Leu, Tyr, Phe, or Met.

In one embodiment, the CDR-H1 may include an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 22, 23, and 24. The CDR-H2 may include an amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 25, and 26. The CDR-H3 may include an amino acid sequence selected from the group consisting of SEQ ID NOS: 3, 27, 28, and 85.

The CDR-L1 may include an amino acid sequence selected from the group consisting of SEQ ID NOS: 10, 29, 30, 31, 32, 33, and 106. The CDR-L2 may include an amino acid sequence selected from the group consisting of SEQ ID NOS: 11, 34, 35, and 36. The CDR-L3 may include an amino acid sequence selected from the group consisting of SEQ ID NOS: 12, 13, 14, 15, 16, 37, 86, and 89.

In another embodiment, the antibody or antigen-binding fragment may comprise or consist essentially of:

a heavy chain variable region comprising a polypeptide (CDR-H1) including an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 22, 23, and 24, a polypeptide (CDR-H2) including an amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 25, and 26, and a polypeptide (CDR-H3) including an amino acid sequence selected from the group consisting of SEQ ID NOS: 3, 27, 28, and 85;

a light chain variable region comprising a polypeptide (CDR-L1) including an amino acid sequence selected from the group consisting of SEQ ID NOS: 10, 29, 30, 31, 32, 33 and 106, a polypeptide (CDR-L2) including an amino acid sequence selected from the group consisting of SEQ ID NOS: 11, 34, 35, and 36, and a polypeptide (CDR-L3) including an amino acid sequence selected from the group consisting of SEQ ID NOS 12, 13, 14, 15, 16, 37, 86, and 89; or

a combination the heavy chain variable region and the light chain variable region.

In one embodiment, the anti-c-Met antibody or antigen-binding fragment may comprise or consist essentially of a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 17, 74, 87, 90, 91, 92, 93, or 94, a light chain variable region comprising the amino acid sequence of SEQ ID NO: 113, 18, 19, 20, 21, 75, 88, 95, 96, 97, 98, 99, or 107, or a combination of the heavy chain variable region and the light chain variable region.

In one embodiment, the anti-c-Met antibody may be a monoclonal antibody. The monoclonal antibody may be produced by the hybridoma cell line deposited with Accession No. KCLRF-BP-00220, which binds specifically to the extracellular region of c-Met protein (refer to Korean Patent Publication No. 2011-0047698, the disclosure of which is incorporated in its entirety herein by reference). The anti-c-Met antibody may include any of the antibodies defined in Korean Patent Publication No. 2011-0047698.

By way of further example, the anti-c-Met antibody or the antibody fragment may include:

a heavy chain including the amino acid sequence selected from the group consisting of the amino acid sequence of SEQ ID NO: 62 (wherein the amino acid sequence from amino acid residues from the 1^(st) to 17^(th) positions is a signal peptide), the amino acid sequence from the 18^(th) to 462^(nd) positions of SEQ ID NO: 62, the amino acid sequence of SEQ ID NO: 64 (wherein the amino acid sequence from the 1^(st) to 17^(th) positions is a signal peptide), the amino acid sequence from the 18^(th) to 461^(st) positions of SEQ ID NO: 64, the amino acid sequence of SEQ ID NO: 66 (wherein the amino acid sequence from the 1^(st) to 17^(th) positions is a signal peptide), and the amino acid sequence from the 18^(th) to 460^(th) positions of SEQ ID NO: 66; and a light chain including the amino acid sequence selected from the group consisting of the amino acid sequence of SEQ ID NO: 68 (wherein the amino acid sequence from the 1^(st) to 20^(th) positions is a signal peptide), the amino acid sequence from the 21^(st) to 240^(th) positions of SEQ ID NO: 68, the amino acid sequence of SEQ ID NO: 70 (wherein the amino acid sequence from the 1^(st) to 20^(th) positions is a signal peptide), the amino acid sequence from the 21^(st) to 240^(th) positions of SEQ ID NO: 70, and the amino acid sequence of SEQ ID NO: 108.

For example, the anti-c-Met antibody may be selected from the group consisting of:

an antibody including a heavy chain including the amino acid sequence of SEQ ID NO: 62 or the amino acid sequence from the 18^(th) to 462^(nd) positions of SEQ ID NO: 62 and a light chain including the amino acid sequence of SEQ ID NO: 68 or the amino acid sequence from the 21^(st) to 240^(th) positions of SEQ ID NO: 68;

an antibody including a heavy chain including the amino acid sequence of SEQ ID NO: 64 or the amino acid sequence from the 18^(th) to 461^(st) positions of SEQ ID NO: 64 and a light chain including the amino acid sequence of SEQ ID NO: 68 or the amino acid sequence from the 21^(st) to 240^(th) positions of SEQ ID NO: 68;

an antibody including a heavy chain including the amino acid sequence of SEQ ID NO: 66 or the amino acid sequence from the 18^(th) to 460^(th) positions of SEQ ID NO: 66 and a light chain including the amino acid sequence of SEQ ID NO: 68 or the amino acid sequence from the 21^(st) to 240^(th) positions of SEQ ID NO: 68;

an antibody including a heavy chain including the amino acid sequence of SEQ ID NO: 62 or the amino acid sequence from the 18^(th) to 462^(nd) positions of SEQ ID NO: 62 and a light chain including the amino acid sequence of SEQ ID NO: 70 or the amino acid sequence from the 21^(st) to 240^(th) positions of SEQ ID NO: 70;

an antibody including a heavy chain including the amino acid sequence of SEQ ID NO: 64 or the amino acid sequence from the 18^(th) to 461^(st) positions of SEQ ID NO: 64 and a light chain including the amino acid sequence of SEQ ID NO: 70 or the amino acid sequence from the 21^(st) to 240^(th) positions of SEQ ID NO: 70;

an antibody including a heavy chain including the amino acid sequence of SEQ ID NO: 66 or the amino acid sequence from the 18^(th) to 460^(th) positions of SEQ ID NO: 66 and a light chain including the amino acid sequence of SEQ ID NO: 70 or the amino acid sequence from the 21^(st) to 240^(th) positions of SEQ ID NO: 70;

an antibody including a heavy chain including the amino acid sequence of SEQ ID NO: 62 or the amino acid sequence from the 18^(th) to 462^(nd) positions of SEQ ID NO: 62 and a light chain including the amino acid sequence of SEQ ID NO: 108;

an antibody including a heavy chain including the amino acid sequence of SEQ ID NO: 64 or the amino acid sequence from the 18^(th) to 461^(st) positions of SEQ ID NO: 64 and a light chain including the amino acid sequence of SEQ ID NO: 108; and

an antibody including a heavy chain including the amino acid sequence of SEQ ID NO: 66 or the amino acid sequence from the 18^(th) to 460^(th) positions of SEQ ID NO: 66 and a light chain including the amino acid sequence of SEQ ID NO: 108.

According to an embodiment, the anti-c-Met antibody may include a heavy chain including the amino acid sequence from the 18^(th) to 460^(th) positions of SEQ ID NO: 66 and a light chain including the sequence from the 21^(st) to 240^(th) positions of SEQ ID NO: 68, or a heavy chain including the amino acid sequence from the 18^(th) to 460^(th) positions of SEQ ID NO: 66 and a light chain including the sequence of SEQ ID NO: 108.

The polypeptide of SEQ ID NO: 70 is a light chain including human kappa (κ) constant region, and the polypeptide with the amino acid sequence of SEQ ID NO: 68 is a polypeptide obtained by replacing histidine at position 62 (corresponding to position 36 of SEQ ID NO: 68 according to kabat numbering) of the polypeptide with the amino acid sequence of SEQ ID NO: 70 with tyrosine. The production yield of the antibodies may be increased by the replacement. The polypeptide with the amino acid sequence of SEQ ID NO: 108 is a polypeptide obtained by replacing serine at position 32 of SEQ ID NO: 108 (corresponding to position 52 of SEQ ID NO: 68, which corresponds to position 27e according to kabat numbering in the amino acid sequence from amino acid residues 21 to 240 of SEQ ID NO: 68; positioned within CDR-L1) with tryptophan. By such replacement, antibodies and antibody fragments including such sequences exhibits increased activities, such as c-Met biding affinity, c-Met degradation activity, Akt phosphorylation inhibition, and the like.

Animal-derived antibodies produced by immunizing non-immune animals with a desired antigen generally invoke immunogenicity when injected to humans for the purpose of medical treatment, and thus chimeric antibodies have been developed to inhibit such immunogenicity. Chimeric antibodies are prepared by replacing constant regions of animal-derived antibodies that cause an anti-isotype response with constant regions of human antibodies by genetic engineering. Chimeric antibodies are considerably improved in an anti-isotype response compared to animal-derived antibodies, but animal-derived amino acids still have variable regions, so that chimeric antibodies have side effects with respect to a potential anti-idiotype response. Humanized antibodies have been developed to reduce such side effects. Humanized antibodies are produced by grafting complementarity determining regions (CDR) which serve an important role in antigen binding in variable regions of chimeric antibodies into a human antibody framework.

The anti c-Met antibodies and antigen-binding fragments thereof may be mouse-derived antibodies, mouse-human chimeric antibodies, humanized antibodies, or human antibodies. The antibodies or antigen-binding fragments thereof may be isolated from a living body or non-naturally occurring. The antibodies or antigen-binding fragments thereof may be recombinant or synthetic. The antibodies may be monoclonal.

An intact antibody includes two full-length light chains and two full-length heavy chains, in which each light chain is linked to a heavy chain by disulfide bonds. The antibody has a heavy chain constant region and a light chain constant region. The heavy chain constant region is of a gamma (γ), mu (μ), alpha (α), delta (δ), or epsilon (ε) type, which may be further categorized as gamma 1 (γ1), gamma 2(γ2), gamma 3(γ3), gamma 4(γ4), alpha 1(α1), or alpha 2(α2). The light chain constant region is of either a kappa (κ) or lambda (λ) type.

As used herein, the term “heavy chain” refers to full-length heavy chain, and fragments thereof, including a variable region V_(H) that includes amino acid sequences sufficient to provide specificity to antigens, and three constant regions, C_(H1), C_(H2), and C_(H3), and a hinge. The term “light chain” refers to a full-length light chain and fragments thereof, including a variable region V_(L) that includes amino acid sequences sufficient to provide specificity to antigens, and a constant region C_(L).

The term “complementarity determining region (CDR)” refers to an amino acid sequence found in a hyper variable region of a heavy chain or a light chain of immunoglobulin. The heavy and light chains may respectively include three CDRs (CDRH1, CDRH2, and CDRH3; and CDRL1, CDRL2, and CDRL3). The CDR may provide contact residues that play an important role in the binding of antibodies to antigens or epitopes. The terms “specifically binding” and “specifically recognized” are well known to one of ordinary skill in the art, and indicate that an antibody and an antigen specifically interact with each other to lead to an immunological activity.

The term “hinge region” as used herein, refers to a region between CH1 and CH2 domains within the heavy chain of an antibody which functions to provide flexibility for the antigen-binding site.

When an animal antibody undergoes a chimerization process, the IgG1 hinge of animal origin is replaced with a human IgG1 hinge or IgG2 hinge while the disulfide bridges between two heavy chains are reduced from three to two in number. In addition, an animal-derived IgG1 hinge is shorter than a human IgG1 hinge. Accordingly, the rigidity of the hinge is changed. Thus, a modification of the hinge region may bring about an improvement in the antigen binding efficiency of the humanized antibody. The modification of the hinge region through amino acid deletion, addition, or substitution is well-known to those skilled in the art.

In one embodiment, the anti-c-Met antibody, or an antigen-binding fragment thereof may be modified by the deletion, insertion, addition, or substitution of at least one amino acid residue on the amino acid sequence of the hinge region so that it exhibit enhanced antigen-binding efficiency. For example, the antibody may include a hinge region including the amino acid sequence of SEQ ID NO: 100(U7-HC6), 101(U6-HC7), 102(U3-HC9), 103(U6-HC8), or 104(U8-HC5), or a hinge region including the amino acid sequence of SEQ ID NO: 105 (non-modified human hinge). In particular, the hinge region has the amino acid sequence of SEQ ID NO: 100 or 101.

In the anti-c-Met antibody, the portion of the light chain and the heavy chain excluding the CDRs, the light chain variable region, and the heavy chain variable region as defined above, for example the light chain constant region and the heavy chain constant region, may be those from any subtype of immunoglobulin (e.g., IgA, IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4), IgM, and the like).

The term “antigen-binding fragment” used herein refers to fragments of an intact immunoglobulin including portions of a polypeptide including at least one CDR or antigen-binding regions having the ability to specifically bind to the antigen. In a particular embodiment, the antigen-binding fragment may be scFv, (scFv)₂, scFvFc, Fab, Fab′, or F(ab′)₂, but is not limited thereto.

Among the antigen-binding fragments, Fab that includes light chain and heavy chain variable regions, a light chain constant region, and a first heavy chain constant region C_(m), has one antigen-binding site.

The Fab′ fragment is different from the Fab fragment, in that Fab′ includes a hinge region with at least one cysteine residue at the C-terminal of C_(H1).

The F(ab′)₂ antibody is formed through disulfide bridging of the cysteine residues in the hinge region of the Fab′ fragment. Fv is the smallest antibody fragment with only a heavy chain variable region and a light chain variable region. Recombination techniques of generating the Fv fragment are widely known in the art.

Two-chain Fv includes a heavy chain variable region and a light chain region which are linked by a non-covalent bond. Single-chain Fv generally includes a heavy chain variable region and a light chain variable region which are linked by a covalent bond via a peptide linker or linked at the C-terminals to have a dimer structure like the two-chain Fv. The peptide linker may be the same as described in the above, for example, those having the amino acid length of about 1 to about 100, about 2 to about 50, particularly about 5 to about 25, and any kinds of amino acids may be included without any restriction.

The antigen-binding fragments may be attainable using protease (for example, the Fab fragment may be obtained by restricted cleavage of a whole antibody with papain, and the F(ab′)₂ fragment may be obtained by cleavage with pepsin), or may be prepared by using a genetic recombination technique.

In the pharmaceutical composition (mixed formulation, first and second pharmaceutical compositions, etc.) or methods described herein, the active ingredients (a c-Met inhibitor or an IGF-1R inhibitor) may be provided (comprised or administered) along with at least one additive selected from the group consisting of a pharmaceutically acceptable carriers, diluents, and excipients.

The pharmaceutically acceptable carrier to be included in the composition or mixed formulation may be those commonly used for the formulation of antibodies, which may be one or more selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginates, gelatin, calcium silicate, micro-crystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, and mineral oil, but are not limited thereto. The pharmaceutical composition may further include one or more selected from the group consisting of a lubricant, a wetting agent, a sweetener, a flavor enhancer, an emulsifying agent, a suspension agent, and preservative.

The pharmaceutical composition or mixed formulation may be administered orally or parenterally. The parenteral administration may include intravenous injection, subcutaneous injection, muscular injection, intraperitoneal injection, endothelial administration, local administration, intranasal administration, intrapulmonary administration, and rectal administration. Since oral administration leads to digestion of proteins or peptides, an active ingredient in the compositions for oral administration must be coated or formulated to prevent digestion in stomach. In addition, the compositions may be administered using an optional device that enables an active substance to be delivered to target cells.

The pharmaceutically effective amount of the pharmaceutical composition, the c-Met inhibitor, or the an IGF-1R inhibitor for a single dose may be prescribed in a variety of ways, depending on factors such as formulation methods, administration manners, age of subjects, body weight, gender, pathologic conditions, diets, administration time, administration interval, administration route, excretion speed, and reaction sensitivity. For example, the pharmaceutically effective amount of the IGF-1R inhibitor for a single dose may be in ranges of about 0.001 to about 100 mg/kg, or about 0.02 to about 10 mg/kg, and the pharmaceutically effective amount of the c-Met inhibitor for a single dose may be in ranges of about 0.001 to 100 mg/kg, or about 0.02 to about 10 mg/kg, but not limited thereto.

The term “pharmaceutically effective amount” used herein refers to an amount of the active ingredient (i.e., the c-Met inhibitor, or the an IGF-1R inhibitor) exhibiting effects in preventing or treating cancer, and may be properly determined in a variety of ways, depending on factors such as formulation methods, administration methods, age of patients, body weight, gender, pathologic conditions, diets, administration time, administration route, excretion speed, and reaction sensitivity.

The pharmaceutically effective amount for the single dose may be formulated into a single formulation in a unit dosage form or formulated in suitably divided dosage forms, or it may be manufactured to be contained in a multiple dosage container. For the kit, the pharmaceutically effective amount of the c-Met inhibitor and the pharmaceutically effective amount of the IGF-1R inhibitor for the single dose (one-time administration) may be each contained in a package container as a base unit.

The administration interval between the co-administrations that is defined as a period between the co-administration and the subsequent co-administration may be, but not limited to, 1-60 seconds, 1-60 minutes, 1-24 hours, 1-7 days, 1-14 days, 7-14 days, 1-30 days, or so on. In case that the co-administration comprises the sequential performance of the first administration step of administering the pharmaceutically effective amount of the c-Met inhibitor and the second administration step of administering the effective amount of the IGF-1R inhibitor, the administration may be simultaneous, or sequential with an interval between the first administration step and the second administration step of about 1 second to about 24 hours, about 1 second to about 12 hours, or about 1 second to about 60 minutes (e.g., about 1 second to about 10 minutes), and their administration may occur in any order.

The mixed formulation or the pharmaceutical compositions for co-administration may be a solution in oil or an aqueous medium, a suspension, a syrup, or an emulsifying solution form, or they may be formulated into a form of an extract, powders, granules, a tablet or a capsule, and they may further include a dispersing agent or a stabilizing agent for their formulation.

In embodiments where the c-Met inhibitor is an anti-c-Met antibody or an antigen binding fragment thereof, the pharmaceutically effective amount of the c-Met inhibitor as an active ingredient may be formulated into an immunoliposome. A liposome containing an antibody may be prepared using any methods well known in the pertinent field. The immunoliposome is a lipid composition including phosphatidylcholine, cholesterol, and polyethyleneglycol-derivated phosphatidylethanolamine, which may be prepared by a reverse phase evaporation method. For example, Fab′ fragments of an antibody may be conjugated to the liposome through a disulfide-exchange reaction. A chemical drug, such as doxorubicin, may further be included in the liposome.

The pharmaceutical composition and method for co-administration proposed in this disclosure can be used for preventing and/or treating a cancer. The cancer may be a cancer related to or characterized by overexpression and/or abnormal activation of c-Met. The cancer may be a solid cancer or blood cancer. The cancer may be a c-Met inhibitor (e.g., an anti-c-Met antibody) non-effective cancer on which a c-Met inhibitor such as an anti-c-Met antibody has no anticancer effect when treated alone (i.e., the cancer is not responsive (susceptive) to treatment with a c-Met inhibitor in the absence of an IGF-1R inhibitor); as well as a cancer on which the c-Met inhibitor has anticancer effect when treated alone or which has no resistance to the c-Met inhibitor. The cancer that is not responsive (susceptive) to treatment with a c-Met inhibitor may have resistance to the c-Met inhibitor in the absence of an IGF-1R inhibitor in the absence of an IGF-1R inhibitor. For instance, the cancer may be, not limited to, at least one selected from the group consisting of squamous cell carcinoma, small-cell lung cancer, non-small-cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung, peritoneal carcinoma, skin cancer, melanoma in the skin or eyeball, rectal cancer, cancer near the anus, esophagus cancer, small intestinal tumor, endocrine gland cancer, parathyroid cancer, adrenal cancer, soft-tissue sarcoma, urethral cancer, chronic or acute leukemia, lymphocytic lymphoma, hepatoma, gastrointestinal cancer, gastric cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular adenoma, breast cancer, colon cancer, large intestine cancer, endometrial carcinoma or uterine carcinoma, salivary gland tumor, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, head and neck cancers, brain cancer, osteosarcoma and so on. In a particular embodiment, the cancer may a cancer having innate or acquired resistance to the c-Met inhibitor. In addition, the cancer may be one selected from gastric cancer, lung cancer, and the like, which have high expression of IGF-1R, but not be limited thereto. The cancer may be a primary cancer or a metastatic cancer.

The prevention and/or treatment effects of the cancer may include effects of not only inhibiting the growth of the cancer cells but also inhibiting the ability of the cancer to migrate, invade healthy cells, and metastasize.

Another embodiment provides partners for combination therapy capable of improving the effect of each other and overcoming resistance to each. In particular, provided is a use of an IGF-1R inhibitor as a partner drug for co-administration with a c-Met inhibitor, that is, the drug is suitable for use in a combination therapy using an c-Met inhibitor, and allows the c-Met inhibitor to exhibit the effect on a disease on which the c-Met inhibitor has no effect when it administered alone or disease having resistance to the c-Met inhibitor.

Therefore, an embodiment provides a pharmaceutical composition for improving the effect of a c-Met inhibitor, which comprises an IGF-1R inhibitor. Another embodiment provides a method for improving the effect of a c-Met inhibitor, comprising co-administering a c-Met inhibitor together with an IGF-1R inhibitor.

The term “improving the effect of a c-Met inhibitor” may comprise making the c-Met inhibitor to have an effect on a disease (e.g., a cancer) on which the c-Met inhibitor has no or only little effect when it is used alone and/or a disease (e.g., a cancer) having resistance to the c-Met inhibitor. Therefore, the composition or method for improving the effect of a c-Met inhibitor may a composition or method for overcoming (treating, reducing, and/or alleviating) resistance to the c-Met inhibitor.

Another embodiment provides a biomarker for predicting and/or monitoring an effect of a c-Met inhibitor, comprising at least one selected from the group consisting of IGF-1R protein and nucleic acids (full length DNA, cDNA, or mRNA) encoding IGF-1R. The nucleic acid encoding IGF-1R may be full length DNA, cDNA, or mRNA of nucleic acids (e.g., full-length DNA, cDNA or mRNA) encoding IGF-1R.

Another embodiment provides a composition or a kit for predicting and/or monitoring an effect of a c-Met inhibitor, comprising a material interacting with at least one selected from the group consisting of IGF-1R protein and nucleic acids encoding IGF-1R.

Another embodiment provides a method for predicting and/or monitoring an effect of a c-Met inhibitor, comprising measuring the level of at least one selected from the group consisting of IGF-1R protein and nucleic acids (full-length DNA, cDNA or mRNA) encoding IGF-1R in a biological sample from a subject (to be tested).

The term “predicting an effect of a c-Met inhibitor” may refer to predicting (determining) whether or not the c-Met inhibitor exhibit its effect on an individual subject by confirming a factor affecting the display of effect of the c-Met inhibitor, such as the presence/absence of innate (inherent) resistance to the c-Met inhibitor. When it is confirmed that a c-Met inhibitor exhibits an effect on an individual subject, the subject may be determined as a subject suitable for application of the c-Met inhibitor. Therefore, the biomarker, composition, kit, or method for predicting an effect of a c-Met inhibitor may be a biomarker, composition, kit, or method for selecting a subject for application of the c-Met inhibitor.

The term “monitoring an effect of a c-Met inhibitor” may refer to monitoring whether the c-Met inhibitor exhibits its effect well or not and/or resistance to the c-Met inhibitor is induced (acquired) by administration of the c-Met inhibitor or not. Therefore, the biomarker, composition, kit, or method for monitoring an effect of a c-Met inhibitor may be a biomarker, composition, kit, or method for monitoring induction (or acquisition) of resistance to the c-Met inhibitor in a subject administered with the c-Met inhibitor.

In an embodiment, provided is a method for predicting an effect of a c-Met inhibitor, selecting a subject for application of a c-Met inhibitor, monitoring an effect of a c-Met inhibitor, or monitoring induction (or acquisition) of resistance to a c-Met inhibitor. In the resistance, since an anti-c-Met antibody is comprised in the c-Met inhibitor, when a resistance to the anti-c-Met antibody exists, the c-Met inhibitor may lose its effect. Therefore, the resistance may refer to a resistance to treatment of a c-Met inhibitor and/or a resistance to treatment of an anti-c-Met antibody.

As described above, a high level of at least one selected from IGF-1R or nucleic acids encoding IGF-1R in a biological sample (e.g., cells, tissues, body fluid, etc.) obtained (separated) from a subject may indicate that the biological sample or the subject has resistance to an anti-c-Met antibody and/or a c-Met inhibitor. Therefore, in the method for predicting an effect of a c-Met inhibitor or selecting a subject for application of a c-Met inhibitor, when at least one selected from IGF-1R or nucleic acids encoding IGF-1R is absent or present at a low level, it can be determined (predicted) that the c-Met inhibitor will exhibit an effect well on the biological sample or the subject from which the biological sample is obtained, or that the biological sample or the subject from which the biological sample is suitable for application of the c-Met inhibitor.

Thus, the method for predicting an effect of a c-Met inhibitor or selecting a subject for application of a c-Met inhibitor may further comprise, after the measuring step, determining (predicting) that the predicting an effect of a c-Met inhibitor, selecting a subject for application of a c-Met inhibitor, or the biological sample or the subject from which the biological sample is suitable for application of the c-Met inhibitor, when at least one selected from IGF-1R or nucleic acids encoding IGF-1R is absent (not detected or not measured) or present at a low level.

As used herein, the term “at least one selected from IGF-1R or nucleic acids encoding IGF-1R is present at a low level” may refer to that the level (amount) of at least one selected from IGF-1R or nucleic acids (e.g., full-length DNA, cDNA or mRNA) encoding IGF-1R in a biological sample (test sample) from a subject (to be tested) is lower than that of a reference sample. The reference sample may refer to a biological sample (e.g., cells, tissues, body fluid, etc.) on which the c-Met inhibitor does not exhibit its effect (for example, an anticancer effect such as inhibiting of cancer cell proliferation, migration (metastasis), or invasion, or inducing cancer cell apoptosis, etc.), or a biological sample (e.g., cells, tissues, body fluid, etc.) separated (obtained) from a subject on which the c-Met inhibitor does not exhibit its effect (for example, an anticancer effect such as inhibiting of cancer cell proliferation, migration (metastasis), or invasion, or inducing cancer cell apoptosis, etc.). For example, the reference sample may be at least one selected from the group consisting of, but not be limited to, H1373 (ATCC, CRL-5866) lung cancer cell line, HCC1806 (ATCC, CRL-2335) breast cancer cell line, Caki-1 (ATCC, HTB-46) renal cancer cell line, SKBR3 (ATCC, HTB-30) breast cancer cell line, BT474 (ATCC, HTB-20) breast cancer cell line, HT-29 (ATCC, HTB-38) colon cancer cell line, LoVo (ATCC, CCL-229) colon cancer cell line, HCT116 (ATCC, CCL-247) colon cancer cell line, SW620 (ATCC, CCL-227) colon cancer cell line, Ls174T (ATCC, CL-188) colon cancer cell line, and cell lines wherein resistance to an anti-c-Met antibody and/or resistance to a c-Met inhibitor is induced by repeated or continued administration of the anti-c-Met antibody and/or c-Met inhibitor.

The method for predicting an effect of a c-Met inhibitor or selecting a subject for application of a c-Met inhibitor may further comprise, after the measuring step and prior to the determining step (if it is comprised), comparing the level (amount) of at least one selected from IGF-1R or nucleic acids encoding IGF-1R in a biological sample (test sample) from a subject of interest (to be tested), with that of a reference sample as described above. For this, the method for predicting an effect of a c-Met inhibitor or selecting a subject for application of a c-Met inhibitor may further comprise, prior to the comparing step, measuring the level (amount) of at least one selected from IGF-1R or nucleic acids (e.g., full-length DNA, cDNA or mRNA) encoding IGF-1R in the reference sample.

The steps of measuring the level of at least one selected from IGF-1R or nucleic acids encoding IGF-1R in the biological sample and the reference sample are performed simultaneously or sequentially in any order.

Alternatively, if the IGF-1R level, which is determined by immunohistochemistry (IHC) using a conventional anti-IGF-1R-antibody (e.g., SAB4300359 (SIGMA), or Gll (Ventana; Catalog Number: 790-4346), etc.), is lower than +1, e.g., 0 or +1, it can be determined that IGF-1R is present at a low level or absent.

The method for predicting an effect of a c-Met inhibitor or selecting a subject for application of a c-Met inhibitor may further comprise, after the step of determining, administering a c-Met inhibitor (e.g., an anti-c-Met antibody or antigen-binding fragment thereof, etc.) to the selected subject, who has low level of IGF-1R as described above.

In another embodiment, a method for monitoring an effect of a c-Met inhibitor, comprising measuring the level (amount) of at least one selected from IGF-1R or nucleic acids (e.g., full-length DNA, cDNA or mRNA) encoding IGF-1R in a biological sample from a subject who is administered with the c-Met inhibitor (or after administration of a c-Met inhibitor). As described above, the method for monitoring an effect of a c-Met inhibitor includes monitoring whether resistance to an anti-c-Met antibody and/or a c-Met inhibitor is induced (acquired) or not.

In the method of monitoring an effect of a c-Met inhibitor, it can be determined that a c-Met inhibitor exhibits its effect (for example, an anticancer effect such as inhibiting of cancer cell proliferation, migration (metastasis), or invasion, or inducing cancer cell apoptosis, etc.) in the subject or resistance to the c-Met inhibitor is not induced in the subject, if the level (amount) of at least one selected from IGF-1R or nucleic acids encoding IGF-1R in a biological sample from the subject who is administered with the c-Met inhibitor (or after administration of a c-Met inhibitor) is equal to (maintained) or decreased compared to that of a biological sample from a subject who is not administered with the c-Met inhibitor (or before administration of a c-Met inhibitor). Alternatively, it can be also determined that a c-Met inhibitor does not exhibit its effect (for example, an anticancer effect such as inhibiting of cancer cell proliferation, migration (metastasis), or invasion, or inducing cancer cell apoptosis, etc.) in the subject or resistance to the c-Met inhibitor is induced in the subject, if the level (amount) of at least one selected from IGF-1R or nucleic acids encoding IGF-1R in a biological sample from the subject who is administered with the c-Met inhibitor (or after administration of a c-Met inhibitor) is increased compared to that of a biological sample from a subject who is not administered with the c-Met inhibitor (or before administration of a c-Met inhibitor).

Therefore, the measuring step of the method of monitoring an effect of a c-Met inhibitor may comprise measuring the levels (amount) of at least one selected from IGF-1R or nucleic acids (e.g., full-length DNA, cDNA or mRNA) encoding IGF-1R in a biological sample from a subject before and after the subject is administered with a c-Met inhibitor.

In addition, the method of monitoring an effect of a c-Met inhibitor may further comprise, after the measuring step,

comparing the levels of at least one selected from IGF-1R or nucleic acids encoding IGF-1R in a biological sample from a subject before and after the subject is administered with a c-Met inhibitor, and/or

determining that 1) the c-Met inhibitor exhibits its effect (for example, an anticancer effect such as inhibiting of cancer cell proliferation, migration (metastasis), or invasion, or inducing cancer cell apoptosis, etc.) in the subject, a resistance to the c-Met inhibitor is not induced (not acquired) in the subject, or the treatment using the c-Met inhibitor can be maintained to the subject, when the level of at least one selected from IGF-1R or nucleic acids encoding IGF-1R in a biological sample from the subject after administration of a c-Met inhibitor is equal to or decreased compared to that of a biological sample from a subject before administration of a c-Met inhibitor, and/or 2) the c-Met inhibitor does not exhibit its effect (for example, an anticancer effect such as inhibiting of cancer cell proliferation, migration (metastasis), or invasion, or inducing cancer cell apoptosis, etc.) in the subject, a resistance to the c-Met inhibitor is induced (acquired) in the subject, or the treatment using the c-Met inhibitor is stopped to the subject, when the level of at least one selected from IGF-1R or nucleic acids encoding IGF-1R in a biological sample from the subject after administration of a c-Met inhibitor is increased compared to that of a biological sample from a subject before administration of a c-Met inhibitor. The c-Met inhibitor may be an anti-c-Met antibody.

The method of monitoring an effect of a c-Met inhibitor may further comprise, after the step of determining, maintaining an administration of a c-Met inhibitor (e.g., an anti-c-Met antibody or antigen-binding fragment thereof, etc.), when it is determined that a c-Met inhibitor exhibits its effect (for example, an anticancer effect such as inhibiting of cancer cell proliferation, migration (metastasis), or invasion, or inducing cancer cell apoptosis, etc.) in the subject or resistance to the c-Met inhibitor is not induced in the subject.

In addition, as described above, when an IGF-1R inhibitor is co-administered with a c-Met inhibitor, the effect of the c-Met inhibitor can be improved (for example, resistance to an anti-c-Met antibody and/or a c-Met inhibitor can be overcome); and thus, even if at least one selected from the group consisting of IGF-1R and a nucleic acid encoding IGF-1R exists in a biological sample from a subject, a desired effect can be achieved by co-administration of an IGF-1R inhibitor and a c-Met inhibitor.

Therefore, another embodiment provides a method for predicting an effect of a combined administration of a c-Met inhibitor and an IGF-1R inhibitor, and/or selecting a patient for application of a combined administration of a c-Met inhibitor and an IGF-1R inhibitor, the method comprising measuring a level of at least one selected from the group consisting of IGF-1R and a nucleic acid encoding IGF-1R in a biological sample from a subject. As described above, the detection (presence) of at least one selected from the group consisting of IGF-1R and a nucleic acid encoding IGF-1R in a biological sample from a subject may indicate that the biological sample or the subject has innate (inherent) or acquired resistance to an anti-c-Met antibody and/or a c-Met inhibitor; however, co-administration of a c-Met inhibitor and an IGF-1R inhibitor can overcome such resistance, thereby achieving an increased therapeutic effect despite such resistance.

Therefore, the method for predicting an effect of a combined administration of a c-Met inhibitor and an IGF-1R inhibitor and/or selecting a subject for application of a combined administration of a c-Met inhibitor and an IGF-1R inhibitor, may further comprise predicting that the combined administration of a c-Met inhibitor and an IGF-1R inhibitor is effective on the biological sample or the subject from which the biological sample is obtained or determining that the biological sample or the subject from which the biological sample is obtained is suitable for application of the combined administration of a c-Met inhibitor and an IGF-1R inhibitor, if at least one selected from the group consisting of IGF-1R and a nucleic acid encoding IGF-1R is detected (present) in a biological sample from a subject. In an embodiment, the biological sample or biological sample or the subject from which the biological sample is obtained may have a resistance to an anti-c-Met antibody and/or a c-Met inhibitor, which is innate (inherently) or acquired by repeated administration of a c-Met inhibitor, wherein such innate (inherently) or acquired resistance to a c-Met inhibitor can be overcome by co-administration of the c-Met inhibitor together with an IGF-1R inhibitor.

The method for predicting an effect of a combined administration of a c-Met inhibitor and an IGF-1R inhibitor and/or selecting a subject for application of a combined administration of a c-Met inhibitor and an IGF-1R inhibitor, may further comprise, after the step of predicting or determining, co-administering a c-Met inhibitor (e.g., an anti-c-Met antibody or antigen-binding fragment thereof, etc.) and an IGF-1R inhibitor to the selected subject.

In the above described method for predicting or monitoring an effect of a c-Met inhibitor or a combined administration of a c-Met inhibitor and an IGF-1R inhibitor or selecting a subject for application of a c-Met inhibitor or a combined administration of a c-Met inhibitor and an IGF-1R inhibitor, the step of measuring the level of at least one selected from IGF-1R or nucleic acids encoding IGF-1R (e.g., the step of measuring the level of at least one selected from IGF-1R or nucleic acids encoding IGF-1R in a biological sample from a subject and a reference sample or in a biological sample from a subject before and after the subject is administered with a c-Met inhibitor may comprise i) applying (adding) an interacting material that interacts with at least one selected from the group consisting IGF-1R or nucleic acids (e.g., full-length DNA, cDNA or mRNA) encoding IGF-1R to the biological sample and/or a reference sample; and ii) quantitatively analyzing the resulting reaction mixture to determine a level (amount) of at least one selected from the group consisting of IGF-1R or nucleic acids (e.g., full-length DNA, cDNA or mRNA) encoding IGF-1R.

In an embodiment, prior to the step i), a step of providing a biological sample (and/or a reference sample) may be further performed. The step of providing a biological sample may comprise obtaining (isolating) a biological sample from a subject or obtaining a biological sample which has been isolated from a subject.

In step i), as will be further elucidated below, the interacting material may be at least one selected from the group consisting of compounds (a small molecular chemical; e.g., general label such as a fluorescent, a dye, etc.), proteins (antibodies, aptamers, etc.), nucleic acids (DNA, RNA, etc.), and the like, binding to IGF-1R or nucleic acids (e.g., full-length DNA, cDNA or mRNA) encoding IGF-1R. For example, the interacting material may be at least one selected from the group consisting of a compound, an antibody, an aptamer, all specifically binding to IGF-1R, and a nucleic acid (e.g., a primer, a probe, an aptamer, etc.) binding to a part or entirety of a nucleic acid (e.g., full-length DNA, cDNA or mRNA) encoding IGF-1R, and optionally, may be conjugated with a label, such as a fluorescent or a dye. The step i) may be configured to form a complex by applying (adding) the interacting material to the biological sample (and/or a reference sample).

In step ii), the reaction mixture may be a complex resulting from interaction (binding) between at least one selected from the group consisting of IGF-1R and a nucleic acid (e.g., full-length DNA, cDNA or mRNA) encoding IGF-1R and the interacting material, which can be obtained in step i). The quantitatively analyzing step may comprise quantifying the complex, the label conjugated to the complex, or the IGF-1R or the nucleic acid gene segregated from the complex after the isolation of the complex from the biological sample (and/or the reference sample). The quantitative analysis of IGF-1R may be performed by any general quantifying means of proteins, such as immunochromatography, immunohistochemistry, immunohistochemical staining, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescence immunoassay (FIA), luminescence immunoassay (LIA), Western blotting, microarray, surface plasmon resonance (SPR), flow cytometry assay (e.g., Cytometric Bead Array (CBA) assay), Intracellular staining, Luminex assay, and the like, but not limited thereto. The quantitative analysis of nucleic acids (e.g., full-length DNA, cDNA or mRNA) encoding IGF-1R may be performed by any general quantifying means of genes (DNA or RNA), such as polymerase chain reaction (PCR; e.g., qPCR, RT-PCR, etc.), FISH (fluorescent in situ hybridization), microarray (e.g., mRNA microarray), and the like, but not limited thereto, which may use the interacting material such as a primer, a probe, or an aptamer, all capable of hybridizing (hybridizable) with a part or entirety of the nucleic acids encoding IGF-1R. In an embodiment, In one embodiment, the primer is designed to detect a fragment of successive base pairs out of the nucleic acid (full-length DNA, cDNA or mRNA) encoding IGF-1R, for example, a fragment of about 5 to about 2000 bp or about 5 to about 1000 bp, e.g., about 10 to about 1500 bp, about 10 to about 1000 bp, about 10 to about 500 bp, about 20 to about 200 bp, or about 50 to about 200 bp, and may be a pair of primers having nucleotide sequences which are respectively hybridizable with (e.g., complementary to) 3′- and 5′-terminal regions of the fragment, the region ranging in size from about 5 to about 100 bp, e.g., about 5 to about 50 bp, about 5 to about 30 bp, or about 10 to about 25 bp (that is, each of the primer pair comprises about 5 to about 100 nt, e.g., about 5 to about 50 nt, about 5 to about 30 nt, or about 10 to about 25 nt). The probe or the aptamer hybridizable with a part or entirety of the nucleic acid encoding IGF-1R may have a nucleotide sequence with a size from about 5 to about 2000 nt, from about 5 to about 1500 nt, from about 5 to about 1000 nt, from about 5 to about 500 nt, from about 5 to about 100 nt, from about 5 to about 50 nt, from about 5 to about 30 nt, or from about 5 to about 25 nt, which is hybridizable with (or complementary to) a fragment of the nucleic acid encoding IGF-1R. As used herein, the term “hybridizable” means pertaining to complementarily binding to a specific region of the nucleic acid, with a sequence complementarity of 80% or higher, e.g., 90% or higher, 95% or higher, 98% or higher, 99% or higher, or 100% between the primer, probe or aptamer and the nucleic acid region.

Considering that characteristic of the therapy using a c-Met inhibitor such as an anti-c-Met antibody, a high level expression of c-Met in a cancer cell can serve as an advantageous condition so that a c-Met inhibitor can more effectively exhibit its effect (e.g., an anticancer effect). Therefore, c-Met and/or nucleic acids (e.g., full-length DNA, cDNA or mRNA) encoding c-Met may be serve as a marker for predicting or monitoring an effect of a c-Met inhibitor or selecting a subject for application of a c-Met inhibitor, and it may be used alone or in combination with IGF-1R and/or nucleic acid encoding IGF-1R.

Therefore, the composition for predicting or monitoring an effect of a c-Met inhibitor or selecting a subject for application of a c-Met inhibitor may further comprise an interacting material that interacts with at least one selected from the group consisting of c-Met and nucleic acids encoding c-Met, in addition to an interacting material that interacts with IGF-1R and/or nucleic acid encoding IGF-1R as described above. The interacting material that interacts with c-Met and/or nucleic acid encoding c-Met may be at least one selected from the group consisting of compounds (a small molecular chemical), proteins (antibodies, aptamers, etc.), nucleic acids (DNA, RNA, etc.), and the like, binding to c-Met or nucleic acids (e.g., full-length DNA, cDNA or mRNA) encoding c-Met. For example, the interacting material may be at least one selected from the group consisting of a compound, an antibody, an aptamer, all specifically binding to c-Met, and a nucleic acid (e.g., a primer, a probe, an aptamer, etc.) binding to a part or entirety of a nucleic acid (e.g., full-length DNA, cDNA or mRNA) encoding c-Met, and optionally, may be conjugated with a label, such as a fluorescent or a dye.

The method for predicting or monitoring an effect of a c-Met inhibitor or selecting a subject for application of a c-Met inhibitor may further comprise measuring a level of c-Met protein or a gene thereof (e.g., full-length DNA, cDNA, mRNA) in the biological sample from a subject. The steps of measuring the level of c-Met and/or c-Met coding nucleic acid and measuring the level of at least one selected from the group consisting of IGF-1R and nucleic acid encoding IGF-1R may be performed simultaneously or sequentially in any order. Details of the measuring step are as described above. For example, a western blotting technique may be employed. In this regard, when a predetermined amount (e.g., about 10 μg) of proteins obtained from a biological sample (e.g., cancer cells or tissues) is loaded on SDS PAGE gel, transferred onto the membrane, and reacted with an anti-c-Met antibody, and then, exposed on ECL reaction for a certain time (e.g., about 30 sec), the detection of a band may indicate that a prerequisite for the c-Met inhibitor therapy is established. In another embodiment, if the c-Met level, which is determined by immunohistochemistry (IHC) using a tissue section (e.g., (Formalin-fixed paraffin-embedded tissues (FFPE), etc.) of a tumor tissue, is +2 or more, the tumor tissue can be determined that a c-Met inhibitor can exhibit its therapeutic efficacy thereon. In another embodiment, when a biological sample is found to have a c-Met mRNA level of about 12,000 or higher, about 13,000 or higher, or about 14,000 or higher, as measured by Affymetrix array (Affymetrix GeneChip Human Genome U133 Plus 2.0 array; using the primer set “203510_at”), a prerequisite for a c-Met inhibitor therapy may be established. Cancer cells characterized by a high expression level of c-Met include cells from lung cancer, breast cancer, brain cancer, stomach cancer, liver cancer, and kidney cancer. However, any cancer cell, although derived from different kinds, may be a target of the c-Met inhibitor therapy if it expresses a high level of c-Met according to personal characteristics of patients.

In another embodiment, a biological sample used in the method for predicting or monitoring the efficacy of a c-Met inhibitor or the method for selecting a subject suitable to the application of a c-Met inhibitor may be a tissue, a cell or body fluid (blood, serum, urine, saliva, etc.) which shows a high expression level of c-Met, for example, a c-Met level of about 12,000 or higher, about 13,000 or higher, or about 14,000 or higher, as measured by Affymetrix array (Affymetrix GeneChip Human Genome U133 Plus 2.0 array; using the primer set “203510_at”).

As used herein, the term “subject (suitable) for the application of a c-Met inhibitor” means a patient to which a c-Met inhibitor therapy is applicable suitably, and the term “subject” may be selected from the group consisting of mammals, such as rodents, e.g., mice, rats, etc., and primates, e.g., humans, monkeys, etc., e.g., a cancer patient. The biological sample may be a patient itself (mammals, such as primates, e.g., humans, monkeys, etc., or rodents, e.g., mice, rats, etc.) or a cell, a tissue or body fluid (e.g., blood, serum, urine, saliva, etc.) isolated from the patient or artificially an artificial culture thereof. The biological sample may be a cell, blood, or a serum.

Another embodiment provides a method of treating and/or preventing a cancer comprising co-administering a c-Met inhibitor to a subject in need thereof. In the method of preventing and/or treating cancer, the subject may be one from whom a biological sample shows a low level (amount) of at least one selected from the group consisting of IGF-1R and a nucleic acid encoding IGF-1R compared to a reference sample, therein the term “low level” and “reference sample” are as described above. For example, the subject may be 1) selected by the above method for selecting a subject for the application of a combined administration of a c-Met inhibitor.

Another embodiment provides a method of treating and/or preventing a cancer comprising co-administering a c-Met inhibitor and an IGF-1R inhibitor to a subject in need thereof. In the method of preventing and/or treating cancer, the subject may be one from whom a biological sample shows the presence of at least one selected from the group consisting of IGF-1R and a nucleic acid encoding IGF-1R. For example, the subject may be selected by the above method for selecting a subject for the application of a combined administration of a c-Met inhibitor and an IGF-1R inhibitor.

The method of treating and/or preventing a cancer may further comprise identifying (selecting) the subject for application of a c-Met inhibitor or application of a combined administration of a c-Met inhibitor and an IGF-1R inhibitor, wherein the detailed process can be performed referring to the method of selecting a subject described above. The c-Met inhibitor may be an anti-c-Met antibody.

In the above methods of treating and/or preventing a cancer, the c-Met inhibitor and/or the IGF-1R inhibitor may be administered at a pharmaceutically effective amount as described above.

For example, the method of treating and/or preventing a cancer may comprise:

identifying a subject for application of a c-Met inhibitor; and

administering a c-Met inhibitor to the subject.

Alternatively, the method of treating and/or preventing a cancer may comprise:

identifying a subject for application of combined administration of a c-Met inhibitor and an IGF-1R inhibitor; and

co-administering a c-Met inhibitor and an IGF-1R inhibitor to the subject.

Alternatively, the method of treating and/or preventing a cancer may comprise:

selecting a subject for application of a c-Met inhibitor by measuring the level of IGF-1R and/or nucleic acid encoding IGF-1R in a biological sample from a subject; and

administering a c-Met inhibitor to the subject.

Alternatively, the method of treating and/or preventing a cancer may comprise:

selecting a subject for application of combined administration of a c-Met inhibitor and an IGF-1R inhibitor by measuring the level of IGF-1R and/or nucleic acid encoding IGF-1R in a biological sample from a subject; and

administering a c-Met inhibitor and an IGF-1R inhibitor to the subject.

In the above methods of treating and/or preventing a cancer, the step of selecting a subject may be performed referring to the above descriptions.

EXAMPLES

Hereafter, the present invention will be described in detail by examples.

The following examples are intended merely to illustrate the invention and are not construed to restrict the invention.

Reference Example 1 Construction of Anti-c-Met Antibody 1.1. Production of “AbF46”, a Mouse Antibody to c-Met

1.1.1. Immunization of Mouse

To obtain immunized mice necessary for the development of a hybridoma cell line, each of five BALB/c mice (Japan SLC, Inc.), 4 to 6 weeks old, was intraperitoneally injected with a mixture of 100 μg of human c-Met/Fc fusion protein (R&D Systems) and one volume of complete Freund's adjuvant. Two weeks after the injection, a second intraperitoneal injection was conducted on the same mice with a mixture of 50 μg of human c-Met/Fc protein and one volume of incomplete Freund's adjuvant. One week after the second immunization, the immune response was finally boosted. Three days later, blood was taken from the tails of the mice and the sera were 1/1000 diluted in PBS and used to examine a titer of antibody to c-Met by ELISA. Mice found to have a sufficient antibody titer were selected for use in the cell fusion process.

1.1.2. Cell Fusion and Production of Hybridoma

Three days before cell fusion, BALB/c mice (Japan SLC, Inc.) were immunized with an intraperitoneal injection of a mixture of 50 μg of human c-Met/Fc fusion protein and one volume of PBS. The immunized mice were anesthetized before excising the spleen from the left half of the body. The spleen was meshed to separate splenocytes which were then suspended in a culture medium (DMEM, GIBCO, Invitrogen). The cell suspension was centrifuged to recover the cell layer. The splenocytes thus obtained (1×10⁸ cells) were mixed with myeloma cells (Sp2/0) (1×10⁸ cells), followed by spinning to give a cell pellet. The cell pellet was slowly suspended, treated with 45% polyethylene glycol (PEG) (1 mL) in DMEM for 1 min at 37° C., and supplemented with 1 mL of DMEM. To the cells was added 10 mL of DMEM over 10 min, after which incubation was conducted in a water bath at 37° C. for 5 min. Then the cell volume was adjusted to 50 mL before centrifugation. The cell pellet thus formed was resuspended at a density of 1˜2×10⁵ cells/mL in a selection medium (HAT medium) and 0.1 mL of the cell suspension was allocated to each well of 96-well plates which were then incubated at 37° C. in a CO₂ incubator to establish a hybridoma cell population.

1.1.3. Selection of Hybridoma Cells Producing Monoclonal Antibodies to c-Met Protein

From the hybridoma cell population established in Reference Example 1.1.2, hybridoma cells which showed a specific response to c-Met protein were screened by ELISA using human c-Met/Fc fusion protein and human Fc protein as antigens.

Human c-Met/Fc fusion protein was seeded in an amount of 50 μL (2 μg/mL)/well to microtiter plates and allowed to adhere to the surface of each well. The antibody that remained unbound was removed by washing. For use in selecting the antibodies that do not bind c-Met but recognize Fc, human Fc protein was attached to the plate surface in the same manner.

The hybridoma cell culture obtained in Reference Example 1.1.2 was added in an amount of 50 μL to each well of the plates and incubated for 1 hour. The cells remaining unreacted were washed out with a sufficient amount of Tris-buffered saline and Tween 20 (TBST). Goat anti-mouse IgG-horseradish peroxidase (HRP) was added to the plates and incubated for 1 hour at room temperature. The plates were washed with a sufficient amount of TBST, followed by reacting the peroxidase with a substrate (OPD). Absorbance at 450 nm was measured on an ELISA reader.

Hybridoma cell lines which secrete antibodies that specifically and strongly bind to human c-Met but not human Fc were selected repeatedly. From the hybridoma cell lines obtained by repeated selection, a single clone producing a monoclonal antibody was finally separated by limiting dilution. The single clone of the hybridoma cell line producing the monoclonal antibody was deposited with the Korean Cell Line Research Foundation, an international depository authority located at Yungun-Dong, Jongno-Gu, Seoul, Korea, on Oct. 6, 2009, with Accession No. KCLRF-BP-00220 according to the Budapest Treaty (refer to Korean Patent Laid-Open Publication No. 2011-0047698).

1.1.4. Production and Purification of Monoclonal Antibody

The hybridoma cell line obtained in Reference Example 1.1.3 was cultured in a serum-free medium, and the monoclonal antibody (AbF46) was produced and purified from the cell culture.

First, the hybridoma cells cultured in 50 mL of a medium (DMEM) supplemented with 10% (v/v) FBS were centrifuged and the cell pellet was washed twice or more with 20 mL of PBS to remove the FBS therefrom. Then, the cells were resuspended in 50 mL of DMEM and incubated for 3 days at 37° C. in a CO₂ incubator.

After the cells were removed by centrifugation, the supernatant was stored at 4° C. before use or immediately used for the separation and purification of the antibody. An AKTA system (GE Healthcare) equipped with an affinity column (Protein G agarose column; Pharmacia, USA) was used to purify the antibody from 50 to 300 mL of the supernatant, followed by concentration with an filter (Amicon). The antibody in PBS was stored before use in the following examples.

1.2. Construction of chAbF46, a Chimeric Antibody to c-Met

A mouse antibody is apt to elicit immunogenicity in humans. To solve this problem, chAbF46, a chimeric antibody, was constructed from the mouse antibody AbF46 produced in Experimental Example 1.1.4 by replacing the constant region, but not the variable region responsible for antibody specificity, with an amino sequence of the human IgG1 antibody.

In this regard, a gene was designed to include the nucleotide sequence of “EcoRI-signal sequence-VH-NheI-CH-TGA-XhoI” (SEQ ID NO: 38) for a heavy chain and the nucleotide sequence of “EcoRI-signal sequence-VL-BsiWI-CL-TGA-XhoI” (SEQ ID NO: 39) for a light chain and synthesized. Then, a DNA fragment having the heavy chain nucleotide sequence (SEQ ID NO: 38) and a DNA fragment having the light chain nucleotide sequence (SEQ ID NO: 39) were digested with EcoRI (NEB, R0101S) and XhoI (NEB, R0146S) before cloning into a pOptiVEC™-TOPO TA Cloning Kit enclosed in an OptiCHO™ Antibody Express Kit (Cat no. 12762-019, Invitrogen), and a pcDNA™3.3-TOPO TA Cloning Kit (Cat no. 8300-01), respectively.

Each of the constructed vectors was amplified using Qiagen Maxiprep kit (Cat no. 12662), and a transient expression was performed using Freestyle™ MAX 293 Expression System (invitrogen). 293 F cells were used for the expression and cultured in FreeStyle™ 293 Expression Medium in a suspension culture manner. At one day before the transient expression, the cells were provided in the concentration of 5×10⁵ cells/ml, and after 24 hours, when the cell number reached to 1×10⁶ cells/ml, the transient expression was performed. A transfection was performed by a liposomal reagent method using Freestyle™ MAX reagent (invitrogen), wherein in a 15 ml tube, the DNA was provided in the mixture ratio of 1:1 (heavy chain DNA:light chain DNA) and mixed with 2 ml of OptiPro™ SFM (invtrogen) (A), and in another 15 ml tube, 100 ul (microliter) of Freestyle™ MAX reagent and 2 ml of OptiPro™ SFM were mixed (B), followed by mixing (A) and (B) and incubating for 15 minutes. The obtained mixture was slowly mixed with the cells provided one day before the transient expression. After completing the transfection, the cells were incubated in 130 rpm incubator for 5 days under the conditions of 37° C. 80% humidity, and 8% CO₂.

Afterwards, the cells were incubated in DMEM supplemented with 10% (v/v) FBS for 5 hours at 37° C. under a 5% CO₂ condition and then in FBS-free DMEM for 48 hours at 37° C. under a 5% CO₂ condition.

After centrifugation, the supernatant was applied to AKTA prime (GE Healthcare) to purify the antibody. In this regard, 100 mL of the supernatant was loaded at a flow rate of 5 mL/min to AKTA Prime equipped with a Protein A column (GE healthcare, 17-0405-03), followed by elution with an IgG elution buffer (Thermo Scientific, 21004). The buffer was exchanged with PBS to purify a chimeric antibody AbF46 (hereinafter referred to as “chAbF46”).

1.3. Construction of Humanized Antibody huAbF46 from Chimeric Antibody chAbF46

1.3.1. Heavy Chain Humanization

To design two domains H1-heavy and H3-heavy, human germline genes which share the highest identity/homology with the VH gene of the mouse antibody AbF46 purified in Reference Example 1.2 were analyzed. An Ig BLAST (www.ncbi.nlm.nih.gov/igblast/) result revealed that VH3-71 has an identity/identity/homology of 83% at the amino acid level. CDR-H1, CDR-H2, and CDR-H3 of the mouse antibody AbF46 were defined according to Kabat numbering. A design was made to introduce the CDR of the mouse antibody AbF46 into the framework of VH3-71. Back mutations to the amino acid sequence of the mouse AbF46 were conducted at positions 30 (S→T), 48 (V→L), 73 (D→N), and 78 (T→L). Then, H1 was further mutated at positions 83 (R→K) and 84 (A→T) to finally establish H1-heavy (SEQ ID NO: 40) and H3-heavy (SEQ ID NO: 41).

For use in designing H4-heavy, human antibody frameworks were analyzed by a BLAST search. The result revealed that the VH3 subtype, known to be most stable, is very similar in framework and sequence to the mouse antibody AbF46. CDR-H1, CDR-H2, and CDR-H3 of the mouse antibody AbF46 were defined according to Kabat numbering and introduced into the VH3 subtype to construct H4-heavy (SEQ ID NO: 42).

1.3.2. Light Chain Humanization

To design two domains H1-light (SEQ ID NO: 43) and H2-light (SEQ ID NO: 44), human germline genes which share the highest identity/homology with the VH gene of the mouse antibody AbF46 were analyzed. An Ig BLAST search result revealed that VK4-1 has a identity/homology of 75% at the amino acid level. CDR-L1, CDR-L2, and CDR-L3 of the mouse antibody AbF46 were defined according to Kabat numbering. A design was made to introduce the CDR of the mouse antibody AbF46 into the framework of VK4-1. Back mutations to the amino acid sequence of the mouse AbF46 were conducted at positions 36 (Y→H), 46 (L→M), and 49 (Y→I). Only one back mutation was conducted at position 49 (Y→I) on H2-light.

To design H3-light (SEQ ID NO: 45), human germline genes which share the highest identity/homology with the VL gene of the mouse antibody AbF46 were analyzed by a search for BLAST. As a result, VK2-40 was selected. VL and VK2-40 of the mouse antibody AbF46 were found to have a identity/homology of 61% at an amino acid level. CDR-L1, CDR-L2, and CDR-L3 of the mouse antibody were defined according to Kabat numbering and introduced into the framework of VK4-1. Back mutations were conducted at positions 36 (Y→H), 46 (L→M), and 49 (Y→I) on H3-light.

For use in designing H4-light (SEQ ID NO: 46), human antibody frameworks were analyzed. A Blast search revealed that the Vk1 subtype, known to be the most stable, is very similar in framework and sequence to the mouse antibody AbF46. CDR-L1, CDR-L2, and CDR-L3 of the mouse antibody AbF46 were defined according to Kabat numbering and introduced into the Vk1 subtype. Back mutations were conducted at positions 36 (Y→H), 46 (L→M), and 49 (Y→I) on H4-light.

Thereafter, DNA fragments having the heavy chain nucleotide sequences (H1-heavy: SEQ ID NO: 47, H3-heavy: SEQ ID NO: 48, H4-heavy: SEQ ID NO: 49) and DNA fragments having the light chain nucleotide sequences (H1-light: SEQ ID NO: 50, H2-light: SEQ ID NO: 51, H3-light: SEQ ID NO: 52, H4-light: SEQ ID NO: 53) were digested with EcoRI (NEB, R0101S) and XhoI (NEB, R0146S) before cloning into a pOptiVEC™-TOPO TA Cloning Kit enclosed in an OptiCHO™ Antibody Express Kit (Cat no. 12762-019, Invitrogen) and a pcDNA™3.3-TOPO TA Cloning Kit (Cat no. 8300-01), respectively, so as to construct recombinant vectors for expressing a humanized antibody.

Each of the constructed vectors was amplified using Qiagen Maxiprep kit (Cat no. 12662), and a transient expression was performed using Freestyle™ MAX 293 Expression System (invitrogen). 293 F cells were used for the expression and cultured in FreeStyle™ 293 Expression Medium in a suspension culture manner. At one day before the transient expression, the cells were provided in the concentration of 5×10⁵ cells/ml, and after 24 hours, when the cell number reached to 1×10⁶ cells/ml, the transient expression was performed. A transfection was performed by a liposomal reagent method using Freestyle™ MAX reagent (invitrogen), wherein in a 15 ml tube, the DNA was provided in the mixture ratio of 1:1 (heavy chain DNA:light chain DNA) and mixed with 2 ml of OptiPro™ SFM (invtrogen) (A), and in another 15 ml tube, 100 ul (microliter) of Freestyle™ MAX reagent and 2 ml of OptiPro™ SFM were mixed (B), followed by mixing (A) and (B) and incubating for 15 minutes. The obtained mixture was slowly mixed with the cells provided one day before the transient expression. After completing the transfection, the cells were incubated in 130 rpm incubator for 5 days under the conditions of 37° C., 80% humidity, and 8% CO₂.

After centrifugation, the supernatant was applied to AKTA prime (GE Healthcare) to purify the antibody. In this regard, 100 mL of the supernatant was loaded at a flow rate of 5 mL/min to AKTA Prime equipped with a Protein A column (GE healthcare, 17-0405-03), followed by elution with an IgG elution buffer (Thermo Scientific, 21004). The buffer was exchanged with PBS to purify a humanized antibody AbF46 (hereinafter referred to as “huAbF46”). The humanized antibody huAbF46 used in the following examples included a combination of H4-heavy (SEQ ID NO: 42) and H4-light (SEQ ID NO: 46).

1.4. Construction of scFv Library of huAbF46 Antibody

For use in constructing an scFv of the huAbF46 antibody from the heavy and light chain variable regions of the huAbF46 antibody, a gene was designed to have the structure of “VH-linker-VL” for each of the heavy and the light chain variable region, with the linker including the amino acid sequence “GLGGLGGGGSGGGGSGGSSGVGS” (SEQ ID NO: 54). A polynucleotide sequence (SEQ ID NO: 55) encoding the designed scFv of huAbF46 was synthesized in Bioneer and an expression vector for the polynucleotide had the nucleotide sequence of SEQ ID NO: 56.

After expression, the product was found to exhibit specificity to c-Met.

1.5. Construction of Library Genes for Affinity Maturation

1.5.1. Selection of Target CDRs and Synthesis of Primers

The affinity maturation of huAbF46 was achieved. First, six complementary determining regions (CDRs) were defined according to Kabat numbering. The CDRs are given in Table 1, below.

TABLE 1 CDR Amino Acid Sequence CDR-H1 DYYMS (SEQ ID NO: 1) CDR-H2 FIRNKANGYTTEYSASVKG (SEQ ID NO: 2) CDR-H3 DNWFAY (SEQ ID NO: 3) CDR-L1 KSSQSLLASGNQNNYLA (SEQ ID NO: 10) CDR-L2 WASTRVS (SEQ ID NO: 11) CDR-L3 QQSYSAPLT (SEQ ID NO: 12)

For use in the introduction of random sequences into the CDRs of the antibody, primers were designed as follows. Conventionally, N codons were utilized to introduce bases at the same ratio (25% A, 25% G, 25% C, 25% T) into desired sites of mutation. In this experiment, the introduction of random bases into the CDRs of huAbF46 was conducted in such a manner that, of the three nucleotides per codon in the wild-type polynucleotide encoding each CDR, the first and second nucleotides conserved over 85% of the entire sequence while the other three nucleotides were introduced at the same percentage (each 5%) and that the same possibility was imparted to the third nucleotide (33% G, 33% C, 33% T).

1.5.2. Construction of a Library of huAbF46 Antibodies and Affinity for c-Met

The construction of antibody gene libraries through the introduction of random sequences was carried out using the primers synthesized in the same manner as in Reference Example 1.5.1. Two PCR products were obtained using a polynucleotide covering the scFV of huAbF46 as a template, and were subjected to overlap extension PCR to give scFv library genes for huAbF46 antibodies in which only desired CDRs were mutated. Libraries targeting each of the six CDRs prepared from the scFV library genes were constructed.

The affinity for c-Met of each library was compared to that of the wildtype. Most libraries were lower in affinity for c-Met, compared to the wild-type. The affinity for c-Met was retained in some mutants.

1.6. Selection of Antibody with Improved Affinity from Libraries

After maturation of the affinity of the constructed libraries for c-Met, the nucleotide sequence of scFv from each clone was analyzed. The nucleotide sequences thus obtained are summarized in Table 2 and were converted into IgG forms. Four antibodies which were respectively produced from clones L3-1, L3-2, L3-3, and L3-5 were used in the subsequent experiments.

TABLE 2 Library Clone constructed CDR Sequence H11-4 CDR-H1 PEYYMS (SEQ ID NO: 22) YC151 CDR-H1 PDYYMS (SEQ ID NO: 23) YC193 CDR-H1 SDYYMS (SEQ ID NO: 24) YC244 CDR-H2 RNNANGNT (SEQ ID NO: 25) YC321 CDR-H2 RNKVNGYT (SEQ ID NO: 26) YC354 CDR-H3 DNWLSY (SEQ ID NO: 27) YC374 CDR-H3 DNWLTY (SEQ ID NO: 28) L1-1 CDR-L1 KSSHSLLASGNQNNYLA  (SEQ ID NO: 29) L1-3 CDR-L1 KSSRSLLSSGNHKNYLA  (SEQ ID NO: 30) L1-4 CDR-L1 KSSKSLLASGNQNNYLA  (SEQ ID NO: 31) L1-12 CDR-L1 KSSRSLLASGNQNNYLA  (SEQ ID NO: 32) L1-22 CDR-L1 KSSHSLLASGNQNNYLA  (SEQ ID NO: 33) L2-9 CDR-L2 WASKRVS (SEQ ID NO: 34) L2-12 CDR-L2 WGSTRVS (SEQ ID NO: 35) L2-16 CDR-L2 WGSTRVP (SEQ ID NO: 36) L3-1 CDR-L3 QQSYSRPYT (SEQ ID NO: 13) L3-2 CDR-L3 GQSYSRPLT (SEQ ID NO: 14) L3-3 CDR-L3 AQSYSHPFS (SEQ ID NO: 15) L3-5 CDR-L3 QQSYSRPFT (SEQ ID NO: 16) L3-32 CDR-L3 QQSYSKPFT (SEQ ID NO: 37)

1.7. Conversion of Selected Antibodies into IgG

Respective polynucleotides encoding heavy chains of the four selected antibodies were designed to have the structure of “EcoRI-signal sequence-VH-NheI-CH-XhoI” (SEQ ID NO: 38). The heavy chains of huAbF46 antibodies were used as they were because their amino acids were not changed during affinity maturation. In the case of the hinge region, however, the U6-HC7 hinge (SEQ ID NO: 57) was employed instead of the hinge of human IgG1. Genes were also designed to have the structure of “EcoRI-signal sequence-VL-BsiWI-CL-XhoI” for the light chain. Polypeptides encoding light chain variable regions of the four antibodies which were selected after the affinity maturation were synthesized in Bioneer. Then, a DNA fragment having the heavy chain nucleotide sequence (SEQ ID NO: 38) and DNA fragments having the light chain nucleotide sequences (DNA fragment including L3-1-derived CDR-L3: SEQ ID NO: 58, DNA fragment including L3-2-derived CDR-L3: SEQ ID NO: 59, DNA fragment including L3-3-derived CDR-L3: SEQ ID NO: 60, and DNA fragment including L3-5-derived CDR-L3: SEQ ID NO: 61) were digested with EcoRI (NEB, R0101S) and XhoI (NEB, R0146S) before cloning into a pOptiVEC™-TOPO TA Cloning Kit enclosed in an OptiCHO™ Antibody Express Kit (Cat no. 12762-019, Invitrogen) and a pcDNA™3.3-TOPO TA Cloning Kit (Cat no. 8300-01), respectively, so as to construct recombinant vectors for expressing affinity-matured antibodies.

Each of the constructed vectors was amplified using Qiagen Maxiprep kit (Cat no. 12662), and a transient expression was performed using Freestyle™ MAX 293 Expression System (invitrogen). 293 F cells were used for the expression and cultured in FreeStyle™ 293 Expression Medium in a suspension culture manner. At one day before the transient expression, the cells were provided in the concentration of 5×10⁵ cells/ml, and after 24 hours, when the cell number reached to 1×10⁶ cells/ml, the transient expression was performed. A transfection was performed by a liposomal reagent method using Freestyle™ MAX reagent (invitrogen), wherein in a 15 ml tube, the DNA was provided in the mixture ratio of 1:1 (heavy chain DNA:light chain DNA) and mixed with 2 ml of OptiPro™ SFM (invtrogen) (A), and in another 15 ml tube, 100 ul (microliter) of Freestyle™ MAX reagent and 2 ml of OptiPro™ SFM were mixed (B), followed by mixing (A) and (B) and incubating for 15 minutes. The obtained mixture was slowly mixed with the cells provided one day before the transient expression. After completing the transfection, the cells were incubated in 130 rpm incubator for 5 days under the conditions of 37° C., 80% humidity, and 8% CO₂.

After centrifugation, the supernatant was applied to AKTA prime (GE Healthcare) to purify the antibody. In this regard, 100 mL of the supernatant was loaded at a flow rate of 5 mL/min to AKTA Prime equipped with a Protein A column (GE healthcare, 17-0405-03), followed by elution with an IgG elution buffer (Thermo Scientific, 21004). The buffer was exchanged with PBS to purify four affinity-matured antibodies (hereinafter referred to as “huAbF46-H4-A1 (L3-1 origin), huAbF46-H4-A2 (L3-2 origin), huAbF46-H4-A3 (L3-3 origin), and huAbF46-H4-A5 (L3-5 origin),” respectively).

1.8. Construction of Constant Region- and/or Hinge Region-Substituted huAbF46-H4-A1

Among the four antibodies selected in Reference Example 1.7, huAbF46-H4-A1 was found to be the highest in affinity for c-Met and the lowest in Akt phosphorylation and c-Met degradation degree. In the antibody, the hinge region, or the constant region and the hinge region, were substituted.

The antibody huAbF46-H4-A1 (U6-HC7) was composed of a heavy chain including the heavy chain variable region of huAbF46-H4-A1, U6-HC7 hinge, and the constant region of human IgG1 constant region, and a light chain including the light chain variable region of huAbF46-H4-A1 and human kappa constant region. The antibody huAbF46-H4-A1 (IgG2 hinge) was composed of a heavy chain including a heavy chain variable region, a human IgG2 hinge region, and a human IgG1 constant region, and a light chain including the light chain variable region of huAbF46-H4-A1 and a human kappa constant region. The antibody huAbF46-H4-A1 (IgG2 Fc) was composed of the heavy chain variable region of huAbF46-H4-A1, a human IgG2 hinge region, and a human IgG2 constant region, and a light chain including the light variable region of huAbF46-H4-A1 and a human kappa constant region. The histidine residue at position 36 on the human kappa constant region of the light chain was changed to tyrosine in all of the three antibodies to increase antibody production.

For use in constructing the three antibodies, a polynucleotide (SEQ ID NO: 63) encoding a polypeptide (SEQ ID NO: 62) composed of the heavy chain variable region of huAbF46-H4-A1, a U6-HC7 hinge region, and a human IgG1 constant region, a polynucleotide (SEQ ID NO: 65) encoding a polypeptide (SEQ ID NO: 64) composed of the heavy chain variable region of huAbF46-H4-A1, a human IgG2 hinge region, and a human IgG1 region, a polynucleotide (SEQ ID NO: 67) encoding a polypeptide (SEQ ID NO: 66) composed of the heavy chain variable region of huAbF46-H4-A1, a human IgG2 region, and a human IgG2 constant region, and a polynucleotide (SEQ ID NO: 69) encoding a polypeptide (SEQ ID NO: 68) composed of the light chain variable region of huAbF46-H4-A1, with a tyrosine residue instead of histidine at position 36, and a human kappa constant region were synthesized in Bioneer. Then, the DNA fragments having heavy chain nucleotide sequences were inserted into a pOptiVEC™-TOPO TA Cloning Kit enclosed in an OptiCHO™ Antibody Express Kit (Cat no. 12762-019, Invitrogen) while DNA fragments having light chain nucleotide sequences were inserted into a pcDNA™3.3-TOPO TA Cloning Kit (Cat no. 8300-01) so as to construct vectors for expressing the antibodies.

Each of the constructed vectors was amplified using Qiagen Maxiprep kit (Cat no. 12662), and a transient expression was performed using Freestyle™ MAX 293 Expression System (invitrogen). 293 F cells were used for the expression and cultured in FreeStyle™ 293 Expression Medium in a suspension culture manner. At one day before the transient expression, the cells were provided in the concentration of 5×10⁵ cells/ml, and after 24 hours, when the cell number reached to 1×10⁶ cells/ml, the transient expression was performed. A transfection was performed by a liposomal reagent method using Freestyle™ MAX reagent (invitrogen), wherein in a 15 ml tube, the DNA was provided in the mixture ratio of 1:1 (heavy chain DNA:light chain DNA) and mixed with 2 ml of OptiPro™ SFM (invtrogen) (A), and in another 15 ml tube, 100 ul (microliter) of Freestyle™ MAX reagent and 2 ml of OptiPro™ SFM were mixed (B), followed by mixing (A) and (B) and incubating for 15 minutes. The obtained mixture was slowly mixed with the cells provided one day before the transient expression. After completing the transfection, the cells were incubated in 130 rpm incubator for 5 days under the conditions of 37° C., 80% humidity, and 8% CO₂.

After centrifugation, the supernatant was applied to AKTA prime (GE Healthcare) to purify the antibody. In this regard, 100 mL of the supernatant was loaded at a flow rate of 5 mL/min to AKTA Prime equipped with a Protein A column (GE healthcare, 17-0405-03), followed by elution with IgG elution buffer (Thermo Scientific, 21004). The buffer was exchanged with PBS to finally purify three antibodies (huAbF46-H4-A1 (U6-HC7), huAbF46-H4-A1 (IgG2 hinge), and huAbF46-H4-A1 (IgG2 Fc)). Among the three antibodies, huAbF46-H4-A1 (IgG2 Fc) was selected for the following examples, and name as L3-1Y-IgG2.

Example 1 Searching Biomarkers for Selecting Anti-c-Met Antibody Effective (Sensitive) Group and Anti-c-Met Antibody Non-Effective (Resistance) Group

Expression levels of receptor tyrosine kinase proteins (RTKs) were measured in cell lines Hs746T(HTB-135, ATCC) and SNU-5(00005, KCLB), on which anti-c-Met antibody L3-1Y/IgG2 exhibits anticancer effect when used alone, and cell lines SNU-668(00668, KCLB) and YCC-11(Yonsei University), on which anti-c-Met antibody L3-1Y/IgG2 does not exhibit anticancer effect when used alone.

In particular, each (4×10⁶ cells) of the cell lines were treated with 10 ug/ml of L3-1Y/IgG2, and then, lysed using lysis buffer 17 (Cat. No. 895943; provided in Human Phospho-RTK Array Kit), to obtain 300 UG of proteins. For comparison, each cell line without antibody treatment was used as a control and subjected to the same experiment as described above. The obtained proteins were diluted with a buffer provided in a kit (R&D ARY001B), incubated at 4° C. for 24 hours, and then analyzed using the chemiluminesnce buffer provided in the kit.

The obtained results are shown in FIG. 1. As shown in FIG. 1, the expression profiles of receptor tyrosine kinase proteins (RTKs) in the above 4 types of cell lines are different from each other. In particular, a considerable difference is observed between protein expression profiles in L3-1Y/IgG2 sensitive groups and L3-1Y/IgG2 resistance groups.

To more clearly confirm the protein expression profiles obtained in FIG. 1, the results of FIG. 1 were measured by Densitometry (GenePix Pro 4.1 software) and classified by the following standards.

(in the above table, the numerical values indicate relative values (intensities) quantified by normalization of darkness and size of dots corresponding to receptor tyrosine kinase proteins (RTKs) with those of positive control dot)

The protein expression levels classified by darkness of dots as above are shown in FIG. 2 (that is, the degree of darkness is directly proportional to the level of protein expression).

To more clearly confirm the protein expression level indicated in FIG. 2, the expression level of c-Met, IGF-1R, EGFR, and ErbB2 were quantified. In particular, pixels of densitometry (GenePix Pro 4.1 software) of dots were measured and a mean pixel density of each dot was calculated by considering the measured value of a positive dot (a control present on each membrane for normalization; Loading control) as 100%. The obtained quantification results are shown in FIG. 3 (c-Met), FIG. 4 (IGF-1R), FIG. 5 (EGFR), and FIG. 6 (ErbB2).

As shown in FIGS. 2-6, IGF-1R is not detected in L3-1Y/IgG2 sensitive groups, whereas it is detected in L3-1Y/IgG2 non-effective (resistance) groups at relatively high level. Therefore, IGF-1R was selected as a biomarker for determining (predicting or screening) effect of an anti-c-Met antibody.

Example 2 Examination of Effect of Co-Administration of an Anti-c-Met Antibody and an IGF-1R Inhibitor in Anti-c-Met Antibody Resistant Cancer Cells

Anti-c-Met antibody resistance acquired gastric cancer cells, MKN45 re#1 and MKN45 re#24, were obtained by long term-administration of anti-c-Met antibody L3-1Y/IgG2 prepared in Reference Example 1 to MKN45 cells (JCRB 0254) on which anti-c-Met antibody L3-1Y/IgG2 exhibits its effect (e.g., cancer cell proliferation inhibition effect) when it is administered alone, and used for the following experiment.

In particular, in order to obtain anti-c-Met antibody resistant MKN45 gastric cancer cells, MKN45 gastric cancer cells were administered with L3-1Y/IgG2 for at least 3 months by increasing the administration concentration. The amount of L3-1Y/IgG2 administered was increased from 1 μg/ml to 10 μg/ml, until resistance to L3-1Y/IgG2 is induced. The obtained L3-1Y/IgG2 resistant clones were named as MKN45re#1 and MKN45re#24, respectively.

In order to confirm the acquisition of resistance to L3-1Y/IgG2, the prepared L3-1Y/IgG2 resistant clones (MKN45 re#1 and MKN45 re#24) and MKN45 with no resistance were administered with various concentrations of L3-1Y/IgG2 (see FIG. 9). 72 hours after the antibody administration, the number of living cells was measured through CellTiter Glo assay (Promega, G7573). The obtained results are shown in FIG. 9. As shown in FIG. 9, the proliferation of MKN45 re#1 and MKN45 re#24 is not inhibited by treatment of L3-1Y/IgG2 even when the concentration of L3-1Y/IgG2 is increased, indicating that MKN45 re#1 and MKN45 re#24 acquire resistance to L3-1Y/IgG2.

The prepared L3-1Y/IgG2 resistance induced cell lines MKN45re#1 and MKN45re#24 were co-administered with L3-1Y/IgG2 and an IGF-1R inhibitor, to examine changes in cell proliferation of the L3-1Y/IgG2 resistance induced cell lines. Each of the L3-1Y/IgG2 resistant MKN45re#1 and MKN45re#24 cell lines was seeded on a 96-well plate at the amount of 10000 cells/well (medium: RPMI (GIBCO); culture conditions: 5% CO₂ and 37° C.), and on the next day, treated with L3-1Y/IgG2 (2 μg/ml) and linsitinib (10 uM) together. 72 hours after the co-treatment, the number of living cells was counted by CellTiter Glo assay (Promega, G7573). For comparison, the same experiments were conducted using a control group with no treatment of L3-1Y/IgG or linsitinib, a group treated with L3-1Y/IgG (2 μg/ml) only, and a group treated with linsitinib (10 uM) only.

The obtained results are shown in FIGS. 7 and 8. As shown in FIGS. 7 and 8, when the L3-1Y/IgG2 resistance-induced MKN45 cell lines MKN45 re#1 and MKN45 re#24 were treated with L3-1Y/IgG2 only or linsitinib only, no cell proliferation inhibition effect is observed, whereas they were treated with L3-1Y/IgG2 and linsitinib together, clear and considerable cell proliferation inhibition effect (30% or more) is observed. These results suggest that by administration of an anti-c-Met antibody (L3-1Y/IgG2) together with an IGF-1R inhibitor (linsitinib), the scope of indications (disease), on which the anti-c-Met antibody can exhibit anticancer effect, can be expanded to those having resistance to the anti-c-Met antibody.

All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

The use of the terms “a” and “an” and “the” and “at least one” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context. 

What is claimed is:
 1. A method of treating a cancer in a subject, or overcoming resistance to a c-Met inhibitor in a subject, the method comprising co-administering a c-Met inhibitor and an insulin-like growth factor 1 receptor (IGF-1R) inhibitor to the subject, wherein the cancer is not susceptive to treatment with a c-Met inhibitor in the absence of an IGF-1R inhibitor.
 2. The method of claim 1, wherein the c-Met inhibitor and an IGF-1R inhibitor are co-administered in a single, mixed formulation, or each of the c-Met inhibitor and an IGF-1R inhibitor are administered in separate compositions, simultaneously or sequentially in any order.
 3. The method of claim 1, wherein the c-Met inhibitor comprises at least one selected from the group consisting of an anti-c-Met antibody or an antigen-binding fragment thereof, an aptamer, siRNA, shRNA, microRNA, a small-molecule c-Met inhibitor or a pharmaceutically acceptable salt thereof.
 4. The method of claim 1, wherein the c-Met inhibitor is an anti-c-Met antibody or an antigen-binding fragment thereof that recognizes or binds to a polypeptide comprising 5 to 19 contiguous amino acid residues within SEQ ID NO: 71 and wherein the polypeptide comprises at least SEQ ID NO:
 73. 5. The method of claim 1, wherein the c-Met inhibitor is an anti-c-Met antibody or an antigen-binding fragment thereof that comprises: (i) heavy chain complementarity determining regions (CDRs) comprising a CDR-H1 comprising SEQ ID NO: 4; a CDR-H2 comprising SEQ ID NO: 2, SEQ ID NO: 5, or an amino acid sequence comprising 8-19 consecutive amino acids within SEQ ID NO: 2 comprising the 3^(rd) to 10^(th) positions of SEQ ID NO: 2; and a CDR-H3 comprising SEQ ID NO: 6, SEQ ID NO: 85, or an amino acid sequence comprising 6-13 consecutive amino acids within SEQ ID NO: 85 comprising the 1^(st) to 6^(th) positions of the amino acid sequence of SEQ ID NO: 85; and (ii) light chain CDRs comprising a CDR-L1 comprising SEQ ID NO: 7, a CDR-L2 comprising SEQ ID NO: 8, and a CDR-L3 comprising SEQ ID NO: 9, SEQ ID NO: 15, SEQ ID NO: 86, or 9-17 consecutive amino acids within SEQ ID NO: 89 comprising the 1^(st) to 9^(th) positions of SEQ ID NO:
 89. 6. The method of claim 5, wherein the anti-c-Met antibody or an antigen-binding fragment thereof comprises: a heavy chain variable region comprising a CDR-H1 comprising SEQ ID NO: 1, 22, 23, or 24, a CDR-H2 comprising SEQ ID NO: 2, 25, or 26, and a CDR-H3 comprising SEQ ID NO: 3, 27, 28, or 85; and a light chain variable region comprising a CDR-L1 comprising SEQ ID NO: 10, 29, 30, 31, 32, 33, or 106, a CDR-L2 comprising SEQ ID NO: 11, 34, 35, or 36, and a CDR-L3 comprising SEQ ID NO: 12, 13, 14, 15, 16, 37, 86, or
 89. 7. The method of claim 5, wherein the anti-c-Met antibody or an antigen-binding fragment thereof comprises: a heavy chain variable region comprising SEQ ID NO: 17, 74, 87, 90, 91, 92, 93, or 94; and a light chain variable region comprising SEQ ID NO: 113, 18, 19, 20, 21, 75, 88, 95, 96, 97, 98, 99, or
 107. 8. The method of claim 5, wherein the anti-c-Met antibody comprises: a heavy chain comprising SEQ ID NO: 62, an amino acid sequence from the 18^(th) to 462^(nd) positions of SEQ ID NO: 62, SEQ ID NO: 64, an amino acid sequence from the 18^(th) to 461^(st) positions of SEQ ID NO: 64, SEQ ID NO: 66, or an amino acid sequence from the 18^(th) to 460^(th) positions of SEQ ID NO: 66; and a light chain comprising SEQ ID NO: 68, an amino acid sequence from the 21^(st) to 240^(th) positions of SEQ ID NO: 68, SEQ ID NO: 70, an amino acid sequence from the 21^(st) to 240^(th) positions of SEQ ID NO: 70, or SEQ ID NO:
 108. 9. The method of claim 1, wherein the c-Met inhibitor comprises at least one selected from the group consisting of crizotinib, cabozantinib, foretinib, PHA-665752, SU11274, SGX-523, PF-04217903, EMD 1214063, golvatinib, INCB28060, MK-2461, tivantinib, NVP-BVU972, AMG458, BMS 794833, BMS 777607, MGCD-265, AMG-208, BMS-754807, JNJ-38877605, and pharmaceutically acceptable salts thereof.
 10. The method of claim 1, wherein the IGF-1R inhibitor comprises at least one selected from the group consisting of an antibody, an aptamer, siRNA, shRNA, microRNA, a small-molecule IGF-1R inhibitor, and a pharmaceutically acceptable salt thereof.
 11. The method of claim 1, wherein the IGF-1R inhibitor comprises at least one selected from the group consisting of linsitinib, NVP-AEW541, GSK1904529A, NVP-ADW742, BMS-536924, figitumumab, cixutumumab, dalotuzumab, R1507, XL-228, INSM-18, BMS-754807, AG-1024, GSK1838705A, PQ 401, and pharmaceutically acceptable salts thereof.
 12. The method of claim 1, wherein the cancer is resistant to treatment with a c-Met inhibitor in the absence of an IGF-1R inhibitor.
 13. The method of claim 12, wherein the cancer is resistant to treatment with an anti-c-Met antibody or antigen-binding fragment thereof in the absence of an IGF-1R inhibitor.
 14. The method of claim 13, wherein the cancer is gastric cancer or lung cancer.
 15. A method of monitoring presence of resistance to a c-Met inhibitor in a subject, or selecting a subject for co-administration of a c-Met inhibitor and an IGF-1R inhibitor, the method comprising measuring level of at least one selected from the group consisting of IGF-1R and a nucleic acid encoding IGF-1R in a biological sample from the subject.
 16. The method of claim 15, further comprising selecting a subject for co-administration of a c-Met inhibitor and an IGF-1R inhibitor if IGF-1R or a nucleic acid encoding IGF-1R is detected in the biological sample.
 17. The method of claim 1, wherein the cancer is characterized by overexpression of c-Met.
 18. A pharmaceutical composition comprising a c-Met inhibitor and an IGF-1R inhibitor. 